KR20230164753A - Perfusion manifold assembly - Google Patents

Abstract

A drop-to-drop connection method is described for installing a microfluidic device in fluid communication with a fluid source or another microfluidic device, such as, but not limited to, installing a microfluidic device in fluid communication with a perfusion manifold assembly. . A microfluidic device detachably connected to the assembly such that fluid enters a port of the microfluidic device from a fluid reservoir at a controllable flow rate, optionally without tubing, such as organ microfluidics on a chip containing cells that mimic cells in an organ in the body. A perfusion manifold assembly allowing perfusion of a device is described.

Description

Perfusion Manifold Assembly {PERFUSION MANIFOLD ASSEMBLY}

A microfluidic device detachably connected to the assembly such that fluid enters a port of the microfluidic device from a fluid reservoir at a controllable flow rate, optionally without tubing, such as mimicking cells in an organ in the body or mimicking at least one function of an organ. A perfusion manifold assembly is contemplated that allows perfusion of organ microfluidic devices on a chip containing cells. Drop-to-Drop (as an embodiment for installing a microfluidic device in fluid communication with a fluid source or another microfluidic device, such as, but not limited to, installing a microfluidic device in fluid communication with a perfusion manifold assembly) A droplet-to-droplet connection method is considered.

Two-dimensional (2D) monolayer cell culture systems have been used in biological research for many years. The most common cell culture platform is two-dimensional (2D) monolayer cell culture in Petri dishes or flasks. Although these 2D in vitro models are less expensive than animal models and contribute to systematic, reproducible, quantitative studies of cell physiology (e.g., in drug discovery and development), the physiology of the information retrieved from in vitro studies to in vivo systems is limited. Academic relevance is often questionable. It is now widely accepted that three-dimensional (3D) cell culture matrices promote several biologically relevant functions that are not observed in 2D monolayer cell cultures. In other words, 2D cell culture systems do not accurately replicate the structure, function, and physiology of living tissues in vivo.

U.S. Patent No. 8,647,861 describes a microfluidic “organ-on-chip” device containing living cells on membranes in microchannels exposed to culture fluid at a predetermined flow rate. In contrast to static 2D cultures, microchannels allow perfusion of cell culture medium through cell cultures during in vitro studies, thus providing a physical environment more similar to in vivo. Briefly, an injection port allows injection of cell culture medium into a cell-containing microfluidic channel or chamber, delivering nutrients and oxygen to the cells. The discharge port then allows the discharge of the remaining medium as well as harmful metabolic by-products.

Although these microfluidic devices are an improvement over traditional static tissue culture models, their small size, scale, and interface make fluid handling difficult. There is a need for a method of controlling perfusion of these devices in such a way that fluid pressure creates a flow rate that applies the desired fluid shear stress to living cells. Ideally, the solution should provide a simple user workflow.

Summary of the invention

The present invention contemplates numerous devices individually and in combination. The present invention relates to one or more microfluidic devices, such as an “organ on a chip” microfluidic device, comprising cells that mimic the function of at least one organ in the body and allowing perfusion and optionally mechanical actuation of the microfluidic device, optionally without tubing. (or simply “microfluidic chip”) a perfusion manifold assembly (also referred to as a cartridge, pod, or perfusion disposable), with or without any requirements or intent for the placement of the components. ) is taken into account. The present invention contemplates numerous embodiments of perfusion manifold assemblies. However, the invention is not intended to be limited to these embodiments. For example, the present invention contemplates combining features from different embodiments (discussed below). Additionally, the present invention contemplates eliminating features from the embodiments (discussed below). Additionally, the present invention contemplates permuting features in embodiments (discussed below).

A culture module is contemplated that allows perfusion and optionally mechanical actuation of one or more microfluidic devices, such as an organ microfluidic device on a chip containing cells that mimic the function of at least one organ in the body. In one embodiment, a microfluidic device includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel. In one embodiment, the culture module includes a pressure manifold that allows perfusion of a microfluidic device, such as an “organ on a chip” microfluidic device that mimics cells in an organ in the body or contains cells that mimic at least one function of an organ. and a fold, optionally maintaining contact with the irrigation disposable and removably connected to the assembly to allow fluid to enter the port of the microfluidic device from the fluid reservoir at a controllable flow rate, optionally without tubing. The perfusion disposables can be used separately from the culture module, and the microfluidic device or chip can be used separately from the perfusion disposables. In one embodiment, the present invention provides a pressure manifold (moving or non-moving) configured to bond with one or more microfluidic devices having an integrated valve capable of preventing gas leaks when not bonded with the microfluidic device. For example, consider any one of the perfusion manifold assembly embodiments described herein).

One embodiment includes a drop-to-drop connection for installing a microfluidic device in fluid communication with a fluid source or another microfluidic device, such as, but not limited to, installing a microfluidic device in fluid communication with a perfusion disposable. is considered. In one embodiment, a microfluidic device includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

A pressure cover is contemplated for pressurizing one or more reservoirs within a perfusion disposable or perfusion manifold assembly (or other microfluidic device), the pressure cover being movable or removably attached to the perfusion disposable or other microfluidic device. , allowing improved access to elements (e.g. storage) from within. The pressure cover can be removed from the perfusion disposable product and the perfusion disposable product can be used without the cover. In one embodiment, a perfusion disposable device includes a microfluidic chip, the chip comprising an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic chip includes cells on a membrane and/or in or on a channel.

A pressure control method is contemplated that allows control of the flow rate (during perfusion of cells) despite the limitations of typical pressure regulators. In one embodiment, the pressure controllers (or actuators) of the culture module are not “on” all the time (or just at one setpoint), but rather they switch in an “on” and “off” pattern (or between two or more settings). do. Accordingly, the switching pattern can be selected such that the average value of the liquid operating pressure in one or more reservoirs of the engaged perfusion disposable product (containing a microfluidic device or chip) corresponds to the desired value. In one embodiment, a microfluidic device includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

In one embodiment, a perfusion manifold assembly comprises: i) a cover or shroud configured to act as a top of one or more fluid reservoirs, ii) one or more fluid reservoirs, iii) a capping layer beneath said fluid reservoir(s), iv ) a fluid backplane comprising a resistor, below and in fluid communication with said fluid reservoir(s), and v) a protruding member or skirt (for engaging the microfluidic device or a carrier containing the microfluidic device). As mentioned above, the cover or shroud can be removed and the perfusion manifold assembly can still be used. In one embodiment, the assembly further includes a fluid port located at the bottom of the fluid backplane. In one embodiment, the capping layer caps the fluid backplane. Without wishing to be bound by theory as to any particular mechanism, these resistors act to stabilize the flow of fluid from the reservoir such that a steady flow can be delivered to the microfluidic device, and/or they act to convert the reservoir pressure into a perfusion flow rate. It is believed that it acts to provide a means for In one embodiment, the lid is maintained on the reservoir using a radial seal. This does not require applied pressure to create a seal. In another embodiment, the lid is retained on the reservoir using one or more clips, screws or other retention mechanisms. In one embodiment, the protruding member or skirt engages a microfluidic chip. In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

In one embodiment, a perfusion manifold assembly includes i) one or more fluid reservoirs, and ii) a fluid backplane comprising a fluid channel terminating in a port and in fluid communication with the fluid reservoir(s). In one embodiment, the fluid backplane includes a resistor. In one embodiment, the perfusion manifold assembly further comprises iii) a protruding member or skirt. In one embodiment, the skirt includes a guiding mechanism (for engaging the microfluidic device or a carrier containing the microfluidic device). In one embodiment, the guide mechanism includes a guide shaft, or a hole, groove, orifice, or other cavity configured to receive the guide shaft. In one embodiment, the guiding mechanism includes a (external or internal) guide track. In one embodiment, the guide track is a side track (for engaging the microfluidic device or carrier). In one embodiment, the perfusion manifold assembly may further include a capping layer that caps the fluid backplane. The above embodiments may optionally further include a cover or lid. In one embodiment, the lid is maintained on the reservoir using a radial seal. This does not require applied pressure to create a seal. In another embodiment, the lid is retained on the reservoir using one or more clips, screws or other retaining mechanisms. In one embodiment, the fluid port is at the bottom of the fluid backplane. In one embodiment, the protruding member or skirt engages a microfluidic chip. In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

In one embodiment, the perfusion manifold assembly includes i) one or more fluid reservoirs, ii) a fluid backplane comprising a resistor below and in fluid communication with the fluid reservoir(s), and iii) (microfluidic device or microfluidic device). and a protruding member or skirt (for engaging a carrier containing the fluidic device). The embodiment may further include a capping layer that caps the fluid backplane. The above embodiments may optionally further include a cover or lid. In one embodiment, the lid is maintained on the reservoir using a radial seal. This does not require applied pressure to create a seal. In another embodiment, the lid is retained on the reservoir using one or more clips, screws or other retaining mechanisms. In one embodiment, the fluid port is at the bottom of the fluid backplane. In one embodiment, the protruding member or skirt engages a microfluidic chip. In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

In one embodiment, a perfusion manifold assembly includes i) one or more fluid reservoirs, ii) a fluid backplane below and in fluid communication with the fluid reservoir(s), and iii) a capping layer capping the fluid backplane. In one embodiment, the fluid backplane includes one or more resistors. In one embodiment, the assembly optionally further comprises iv) a protruding member or skirt (for engaging the microfluidic device or a carrier containing the microfluidic device). The above embodiments may optionally further include a cover or lid. In some embodiments, attachment of the microfluidic device and perfusion disposable is through engagement with the skirt. However, in other embodiments, attachment is accomplished directly with the assembly (without a skirt or other external extension). In one embodiment, the protruding member or skirt engages a microfluidic chip. In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

In one embodiment, the invention provides a fluid backplane comprising i) one or more fluid reservoirs, ii) a fluid channel terminating in a port and a fluid resistor, and located below and in fluid communication with said fluid reservoir, and iii) 1 Consider a flow-through manifold assembly comprising a protruding member or skirt having more than two side tracks. In one embodiment, the port is located at the bottom of the fluid backplane. In one embodiment, the one or more side tracks are configured to engage a microfluidic device located on a microfluidic device carrier having one or more outer edges configured to slidably engage the one or more side tracks. In one embodiment of the slideable engagement, the connection approach to the perfusion manifold includes 1) a sliding action, 2) a pivot movement, and 3) a snap fit to provide alignment and fluid connection in a single action. . 1) In the sliding step, the chip (or other microfluidic device) is placed into a sliding carrier to align the fluid ports. 2) In the pivot step, the carrier and chip (or other microfluidic device) are pivoted until the ports are in contact with the fluid. 3) At the clip or snap fit stage, the necessary force is provided to provide a positive seal. In one embodiment, the protruding member or skirt engages a microfluidic chip. In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

In one embodiment, the carrier has a cutout or “window” (e.g., a transparent window) for imaging (e.g., using a microscope) the cells within the microfluidic chip. In one embodiment, there is a corresponding cutout or window (eg, clear) on the perfusion disposable. In one embodiment, the microfluidic device includes features of the carrier to eliminate the need for a separate substrate. In one embodiment, a microfluidic device includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

In one embodiment, the invention provides a fluid backplane comprising i) one or more fluid reservoirs, ii) a fluid channel terminating in a protruding member or skirt and a fluid resistor, iii) positioned below and in fluid communication with said fluid reservoir, iii) ) Consider a perfusion manifold assembly comprising a protruding member or skirt having one or more fluid ports and one or more side tracks. In one embodiment, the one or more side tracks are configured to engage a microfluidic device located on a microfluidic device carrier having one or more outer edges configured to slidably engage the one or more side tracks. In one embodiment of slidable engagement, the connection approach to the perfusion manifold includes 1) a sliding action, 2) a pivot movement, and 3) a snap fit to provide alignment and fluid connection in a single action. 1) In the sliding step, the chip (or other microfluidic device) is placed in a sliding carrier to align the fluid ports. 2) In the pivot step, the carrier and chip (or other microfluidic device) are pivoted until the ports are in contact with the fluid. 3) At the clip or snap fit stage, the necessary force is provided to provide a positive seal. In one embodiment, the microfluidic device includes features of a carrier to eliminate the need for a separate substrate. In one embodiment, the carrier has a cutout or “window” (e.g., a transparent window) for imaging (e.g., using a microscope). In one embodiment, there is a cutout or window (e.g., clear) corresponding to the perfusion disposable (e.g., in the fluid layer). In one embodiment, the present invention contemplates control of the focal plane position and alignment (flatness relative to the microscope stage) on which the chip is placed. It is desirable to minimize the working distance required for imaging (since the longer the working distance, the greater the strain on the objective lens). The invention is not intended to be limited by imaging approach, and imaging may be straight (objective from above) or inverted (objective from below). Although certain embodiments have cutouts or windows on only one side for a particular imaging modality (e.g., epifluorescence), in preferred embodiments the present invention contemplates cutouts or windows on both sides of the chip to enable transmitted light imaging. do. In one embodiment, the resistor includes a tortuous channel. In one embodiment, the fluid backplane is made of Cyclo Olefin Polymer (COP) (e.g., commercially available Zeonor 1420R) and has linear fluid channels in fluid communication with the tortuous channels. and the linear channel terminates in one or more ports. In one embodiment, the skirt is made of polycarbonate (PC). In one embodiment, the assembly further includes a cover for the fluid reservoir, the cover including a plurality of ports optionally associated with a strainer. In some embodiments, the cover port includes a through-hole and the strainer is positioned over a corresponding hole in the gasket. In some embodiments, the cover includes one or more channels passing through one or more of the ports (so that the ports are not simply through-holes). In one embodiment, the side track includes a closed first end proximate the reservoir and an open second end distal the reservoir, wherein the open end is connected to the one or more of the microfluidic device carriers. Includes an angled slide for engagement with the outer edge. In one embodiment, the side track includes a linear region between the closed first end and the open second end. In one embodiment, the protruding member or skirt engages a microfluidic chip. In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

The present invention also contemplates systems that include a perfusion manifold assembly. In one embodiment, the present invention provides a perfusion manifold comprising: a) i) one or more fluid reservoirs, ii) a fluid backplane positioned below and in fluid communication with said fluid reservoirs, and iii) a skirt or other protruding member. assembly; and b) a microfluidic device or chip engaging a perfusion manifold assembly through the skirt. In one embodiment, the microfluidic device is engaged in a removable manner. In one embodiment, the microfluidic device is connected in a non-removable manner (e.g., one-time connection), whether through a locking mechanism or using an adhesive (e.g., an adhesive layer to assist in the quality of fluid sealing). ) are interlocked. In one embodiment, the skirt has a guiding mechanism for engaging the microfluidic device. In one embodiment, the guide mechanism includes a guide shaft, or a hole, groove, orifice, or other cavity configured to receive the guide shaft. In one embodiment, the guiding mechanism comprises a guide track (external or internal). In one embodiment, the guide track is a side track. In one embodiment, the microfluidic device or chip is within a carrier, which engages a perfusion manifold assembly through the side tracks of the skirt. In one embodiment, the microfluidic device has one or more features of the carrier to eliminate the need for an additional substrate, such as a carrier. In one embodiment, a microfluidic device includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel. In one embodiment, the assembly further includes a cover or cover assembly for the fluid reservoir, the cover including a plurality of ports optionally associated with a strainer. In some embodiments, the cover port includes a through-hole and the strainer is positioned over a corresponding hole in the gasket. In some embodiments, the cover includes one or more channels passing through one or more of the ports (so that the ports are not simply through-holes).

In one embodiment, the invention comprises a) i) one or more fluid reservoirs, ii) a fluid channel terminating in a fluid discharge port at the bottom of the fluid backplane and a fluid resistor positioned below the fluid reservoir to provide fluid therewith. a flow manifold assembly comprising a fluid backplane in communication, and iii) a skirt or other protruding member having one or more side tracks; and b) a microfluidic device positioned on a carrier having at least one outer edge releasably engaged with the one or more side tracks of the skirt, wherein the microfluidic device has iii) a mating surface. ) on ii) i) microchannels in fluid communication with the perfusion manifold assembly through one or more injection ports, wherein fluid flows from the fluid reservoir of the perfusion manifold assembly through the one or more fluid discharge ports. wherein the one or more fluid injection ports of the microfluidic device are positioned relative to the one or more fluid discharge ports of the perfusion manifold assembly, under conditions allowing flow to the one or more fluid injection ports of the microfluidic device. Consider the system. In one embodiment, the carrier is engaged in a removable manner. In one embodiment, the carrier is engaged in a non-removable manner (e.g., a one-time connection), whether through a locking mechanism or using an adhesive (e.g., an adhesive layer to assist in the quality of fluid sealing). all. In one embodiment, a microfluidic device includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel. In one embodiment, the assembly further includes a cover for the fluid reservoir, the cover including a plurality of openings associated with the channel. In one embodiment, the assembly further includes a cover for the fluid reservoir, the cover including a plurality of ports optionally associated with a strainer. In some embodiments, the cover port includes a through-hole and the strainer is positioned over a corresponding hole in the gasket. In some embodiments, the cover includes one or more channels passing through one or more of the ports (so that the ports are not simply through-holes).

In one embodiment, the present invention provides a device comprising: a) i) one or more fluid reservoirs, ii) a fluid backplane comprising a fluid channel terminating in a skirt and a fluid resistor, and iii) located below and in fluid communication with said fluid reservoir; A perfusion manifold assembly including a skirt having one or more fluid outlet ports and one or more side tracks; and b) a microfluidic device positioned on a carrier having at least one outer edge releasably engaging with said one or more side tracks of said skirt, said microfluidic device iii) on a bonding surface ii) i) a microchannel in fluid communication with the perfusion manifold assembly through one or more injection ports, wherein fluid flows from the fluid reservoir of the perfusion manifold assembly to the microfluidic device through the one or more fluid discharge ports. The system wherein the one or more fluid injection ports of the microfluidic device are positioned relative to the one or more fluid discharge ports of the skirt of the perfusion manifold assembly, under conditions allowing flow to the one or more fluid injection ports. Consider. In one embodiment, a microfluidic device includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel. In a preferred embodiment, the microfluidic device comprises living cells perfused with fluid from the fluid reservoir. In one embodiment, the assembly further includes a cover for the fluid reservoir, the cover including a plurality of ports optionally associated with a strainer. In some embodiments, the cover port includes a through-hole and the strainer is positioned over a corresponding hole in the gasket. In some embodiments, the cover includes one or more channels passing through one or more of the ports (so that the ports are not simply through-holes).

In a particularly preferred embodiment, the microfluidic device or chip (whether located in a carrier or not) comprises at least two different cell types that function together in a manner that mimics the function of one or more cells in an organ of the body. In one embodiment, the microfluidic device includes a membrane having an upper and lower surface, wherein the upper surface comprises a first cell type and the lower surface comprises a second cell type. In one embodiment, a microfluidic device includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the first cell type is an epithelial cell and the second cell type is an endothelial cell. In preferred embodiments, the membrane is porous (e.g., porous to fluids, gases, cytokines and other molecules, and in some embodiments, porous to cells, allowing cells to migrate through the membrane).

In one embodiment, the invention provides a stand having a) a portion configured to receive i) a chip at least partially contained in a carrier, ii) cells, iii) a seeding guide and iv) at least one seeding guide in a stable mounting position. providing steps; b) engaging the seeding guide with the carrier, thereby creating an engaged seeding guide; c) mounting the engaged seeding guide on the stand, and d) seeding the cells onto the chip while the seeding guide is in a stable mounting position. , having a port associated with one or more microfluidic channels). In one embodiment, the seeding guide is configured to engage (eg, using a guide track) an edge of the carrier. In one embodiment, the seeding guide includes side tracks (similar or identical to those on the skirt of one embodiment of a perfusion manifold assembly) for engaging an edge of the carrier. In one embodiment of this method, multiple seeding guides are mounted on a stand to allow multiple chips to be seeded with cells. In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic chip after said seeding comprises cells on the membrane and/or in or on the channels. In one embodiment, the method comprises, after the seeding of step d), e) separating the carrier from the seeding guide, and f) attaching the perfusion manifold assembly to the carrier comprising the microfluidic chip comprising cells. It additionally includes a step of engaging.

In one embodiment, the present invention provides a stand comprising: a) a stand having a portion configured to receive i) a chip at least partially contained in a seeding guide, ii) a cell, and iii) at least one seeding guide in a stable mounting position; b) engaging the stand with the seeding guide; and c) seeding the cells on the chip while the seeding guide is in a stable mounting position. Consider seeding methods. In one embodiment of this method, multiple seeding guides engage the stand to allow multiple chips to be seeded with cells. In one embodiment of this method, there is no chip carrier. In another embodiment, the chip carrier acts as a seeding guide (without a separate seeding guide engaging the carrier).

In a preferred embodiment, when the one or more fluid injection ports of the microfluidic device are positioned relative to the one or more fluid outlet ports of the perfusion manifold assembly, the carrier has a locking mechanism to limit movement of the carrier. Includes additional The invention is not intended to be limited to the nature of the locking mechanism. In one embodiment, the locking mechanism is selected from the group consisting of clips, clamps, studs, and screws. In one embodiment, the locking mechanism engages with a friction fit. The locking mechanism may allow for releasable engagement or non-removable engagement.

The present invention also contemplates methods of perfusing cells using a perfusion manifold assembly. In one embodiment, the invention provides a device comprising: A) a) i) one or more fluid reservoirs, ii) a fluid backplane below and in fluid communication with said fluid reservoirs and comprising a fluid channel terminating in an outlet port, and iii) ) a perfusion manifold assembly including a skirt or other protruding member containing a guiding mechanism; and b) a microfluidic device positioned on a carrier configured to engage with the guide mechanism of the skirt, comprising i) a living cell, and ii) a microchannel in fluid communication with ii) one or more injection ports on the bonding surface. providing steps; B) positioning the carrier to engage the guide mechanism of the skirt; and C) the microfluidic device is connected such that fluid flows from the fluid reservoir of the perfusion manifold assembly through the one or more fluid discharge ports to the one or more fluid injection ports and the microchannel of the microfluidic device. Perfusing the cells by moving the carrier until the one or more fluid injection ports of the microfluidic device are positioned relative to the one or more fluid outlet ports of the perfusion manifold assembly under conditions that: Consider a method of perfusing cells. In one embodiment, the fluid backplane includes a fluid resistor. In one embodiment, the guide mechanism includes a guide shaft, or a hole, groove, orifice, or other cavity configured to receive the guide shaft. In one embodiment, the guiding mechanism includes a guide track (external or internal). In one embodiment, the guide track is a side track. In one embodiment, the carrier includes one or more outer edges configured to engage the one or more side tracks of the skirt. In one embodiment, the movement of step C) comprises sliding the carrier along the side track until the injection and discharge ports are positioned relative to each other. In one embodiment, the one or more injection ports on the bonding surface of the microfluidic device comprise a droplet that protrudes above the bonding surface, and the one or more outlet ports on the perfusion manifold comprise a projecting droplet, comprising: Let the sliding of C) cause a point-to-point connection. In one embodiment, the carrier is engaged in a removable manner. In another embodiment, the carriers are engaged in a non-removable manner (eg, one-time connection). In one embodiment, the assembly further includes a cover or shroud for the fluid reservoir, the cover including a plurality of ports optionally associated with a strainer. In some embodiments, the cover port includes a through-hole and the strainer is positioned over a corresponding hole in the gasket. In some embodiments, the cover includes one or more channels passing through one or more of the ports (so that the ports are not simply through-holes).

In one embodiment, the present invention provides a device comprising: A) a) i) one or more fluid reservoirs, ii) a fluid backplane comprising a fluid channel terminating in a skirt and a fluid resistor, the fluid backplane being located underneath and in fluid communication with said fluid reservoir; iii) a perfusion manifold assembly comprising a skirt having one or more fluid outlet ports and one or more side tracks; and b) one configured to detachably engage with said one or more side tracks of said skirt, comprising i) living cells, and ii) microchannels in fluid communication with ii) one or more injection ports on iii) an adhesive surface. Providing a microfluidic device positioned on a carrier having at least an outer edge; B) positioning the carrier such that the at least one outer edge engages the at least one side track of the skirt; and C) the microfluidic device is connected such that fluid flows from the fluid reservoir of the perfusion manifold assembly through the one or more fluid discharge ports to the one or more fluid injection ports and the microchannel of the microfluidic device. sliding the carrier along the side track until the one or more fluid injection ports of the microfluidic device are positioned relative to the one or more fluid discharge ports of the skirt of the perfusion manifold assembly, A method of perfusing a cell is contemplated, comprising perfusing the cell. In one embodiment, the one or more injection ports on the bonding surface of the microfluidic device comprise a protruding droplet above the bonding surface, and the one or more discharge ports on the skirt include a protruding droplet, wherein the microfluidic device The sliding of step C) causes a drop-to-drop connection when the one or more fluid injection ports of the device are positioned relative to the one or more fluid discharge ports of the skirt of the perfusion manifold assembly.

In one embodiment, the drop-to-drop connection does not allow air to enter one or more fluid injection ports. In one embodiment, the bonding surface proximate to the droplet is hydrophobic.

In one embodiment, the method further comprises activating a locking mechanism to limit movement of the carrier. In one embodiment, the method further comprises installing the perfusion manifold assembly in an incubator with the connected microfluidic device.

In one embodiment, the method (described for any of the embodiments of the perfusion method above) further comprises installing the perfusion manifold assembly on, in, or in contact with a culture module with the connected microfluidic device. Included as. In one embodiment, the fluid reservoir of the perfusion manifold assembly is covered with a cover assembly comprising a cover having a plurality of ports, the culture module comprising an abutment surface having pressure points corresponding to the ports on the cover, Installing the perfusion manifold assembly with the associated microfluidic device within or on the culture module causes contact of the port with the pressure point. In one embodiment, the fluid reservoir of the perfusion manifold assembly is covered with a cover assembly comprising a cover having a plurality of ports, the culture module comprising an abutment surface having pressure points corresponding to the ports on the cover, After installing the perfusion manifold assembly in or on the culture module with the connected microfluidic device, a pressure point on the mating surface of the culture module comes into contact with the through-hole of the cover assembly. In one embodiment, the fluid reservoir of the perfusion manifold assembly is covered with a cover assembly comprising a cover having a filter and a plurality of through-hole ports associated with corresponding holes in the gasket, and the culture module is located on the cover. Installing the perfusion manifold assembly with the associated microfluidic device on the culture module, including an abutment surface having a pressure point corresponding to the through-hole port, ensures contact of the through-hole with the pressure point. wake up In one embodiment, the fluid reservoir of the perfusion manifold assembly is covered with a cover assembly comprising a cover having a plurality of through-hole ports associated with corresponding holes in a filter and a gasket, and the culture module is positioned in the through-hole port on the cover. - pressure points of a bonding surface of a culture module after installing said perfusion manifold assembly in or on said culture module with said connected microfluidic device, including a bonding surface having a pressure point corresponding to an orifice port. This comes into contact with the through-hole of the cover assembly.

In one embodiment, the culture module includes a volumetric controller. In one embodiment, the volumetric controller applies pressure to the fluid reservoir through the pressure point corresponding to the port on the cover. In one embodiment, the culture module includes a pressure actuator. In one embodiment, the culture module includes a pressure controller. In one embodiment, the pressure controller applies pressure to the fluid reservoir through the pressure point (e.g., on a pressure manifold) that corresponds to the port (e.g., through-hole port) on the cover. . In one embodiment, the culture module includes multiple perfusion manifold assemblies. In one embodiment, the culture module includes an integrated valve. In one embodiment, the integrated valve is in a pressure manifold. In one embodiment, the valve comprises a Schrader valve.

The present invention also contemplates a culture module as a device. In one embodiment, the device includes an actuation assembly configured to move a plurality of microfluidic devices (e.g., a perfusion manifold assembly described herein) relative to a pressure manifold that includes integrated valves. In one embodiment, it is configured to move the microfluidic device upward relative to a non-moving pressure manifold. In one embodiment, the device includes an actuation assembly configured to move one or more perfusion manifold assemblies into contact with a pressure manifold. In one embodiment, the device includes an actuation assembly configured to move a pressure manifold (up or down) into contact with a plurality of perfusion manifold assemblies. In some embodiments, the pressure manifold includes an integrated valve and an elastomeric membrane. In some embodiments, the resilient/flexible seal is disposed on the pod or lid rather than on the pressure manifold. In other embodiments, the invention is not considered to be limited to membranes because the membranes are described in only one particular way; In other embodiments, o-rings, gaskets (thicker than the membrane), flexible materials, or vacuum grease are used instead. In one embodiment, the valve comprises a Schrader valve. In some embodiments, the pressure manifold is adapted to sense the presence of a coupled perfusion manifold assembly or microfluidic device, for example, to reduce leakage of pressure or fluid in the absence of the coupled device. Importantly, in a preferred embodiment, the pressure manifold takes several pressure sources and distributes them to all perfusion manifold assemblies. In some embodiments, the pressure manifold is also designed to align directly with the flow manifold assembly (e.g., via alignment features at the pressure manifold mating surface). In one embodiment, the perfusion manifold assembly slides with an alignment feature at the bottom of the pressure manifold to ensure that the seal in the pressure manifold is always aligned with the ports on the perfusion manifold assembly. In some embodiments, when actuating the pressure manifold, the pressure manifold has a set of springs that press down on the perfusion manifold assembly. These springs push the cover against a reservoir in the perfusion manifold assembly, creating a seal that maintains pressure (avoiding leaks) within the perfusion manifold assembly as pressure passes through the cover ports.

The present invention also contemplates culture modules and perfusion disposables (PDs) as systems. In one embodiment, the system includes a device that includes an actuating assembly configured to move a plurality of microfluidic devices (e.g., a perfusion manifold assembly described herein) relative to a pressure manifold that includes integrated valves. In one embodiment, it is configured to move the microfluidic device upward relative to a non-moving pressure manifold. In one embodiment, the system includes a device comprising a) an actuation assembly configured to b) move a plurality of microfluidic devices (e.g., perfusion disposables) into contact with a pressure manifold. In one embodiment, the system comprises a) a device comprising an actuating assembly configured to move a pressure manifold comprising an integrated valve and seal (e.g., an elastomeric membrane), and b) the seal (e.g., an elastomeric membrane). For example, an elastomeric membrane). In one embodiment, the microfluidic device is a perfusion disposable device. In some embodiments, the resilient/flexible seal is disposed on the pod or lid rather than on the pressure manifold. In other embodiments, the invention is not considered to be limited to membranes because the membranes are described in only one particular way; In other embodiments, o-rings, gaskets (thicker than the membrane), flexible materials, or vacuum grease are used instead. In one embodiment, the valve comprises a Schrader valve. In one embodiment, the manifold uses a dual-stable engagement mechanism so that the actuator does not always have to provide engagement and continuous pressure against the cover. In a dual-stable mechanism, the actuator may engage the manifold and then turn off. This is useful in situations where the actuator generates excessive heat while providing power for long periods of time. In one embodiment, the perfusion disposable is engaged with a microfluidic chip. In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

The invention also provides a drop-to-drop connection for installing a microfluidic device in fluid communication with a fluid source or another microfluidic device, such as, but not limited to, installing a microfluidic device in fluid communication with a perfusion manifold assembly. Consider the method. In one embodiment, the invention is a fluidic device comprising a substrate having a first surface comprising one or more fluid ports, wherein the first surface comprises one or more liquids comprising a first liquid in the one or more fluid ports. Consider a fluidic device adapted to stably retain liquid droplets. In one embodiment, the first surface includes one or more regions surrounding one or more fluid ports, the regions adapted to resist wetting by the first liquid. In one embodiment, the region is adapted to be hydrophobic. In one embodiment, the one or more regions comprise a first material selected to resist wetting by the first liquid. It is not intended that the invention be limited to any particular first material. However, in one embodiment, the first material is poly-tetrafluoroethylene (PTFE), perfluoroalkoxy alkane (PFA), fluorinated ethylenepropylene (FEP), polydimethylsiloxane (PDMS), nylon (some grades are some are hydrophilic and some are hydrophobic), polypropylene, polystyrene and polyimide. In one embodiment, the substrate includes the first material. In one embodiment, the first material is bonded, attached, coated, or sputtered onto the first surface. In one embodiment, the first material includes a hydrophobic gasket. In one embodiment, the one or more regions are adapted to resist wetting by the first liquid by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents. do.

In one embodiment, the first surface includes one or more regions surrounding one or more fluid ports, the regions adapted to promote wetting by the first liquid. In one embodiment, the region is adapted to be hydrophilic. In one embodiment, the one or more regions comprise a first material selected to promote wetting by the first liquid. Again, it is not intended that the invention be limited to any particular first material. However, in one embodiment, the first material is polymethylmethacrylate (PMMA), polyvinyl alcohol (PVOH), polycarbonate (PC), polyether ether ketone (PEEK), polyethylene terephthalate (PET), A group consisting of polyfulphone, polystyrene, polyvinyl acetate (PVA), nylon, polyvinyl fluoride (PVF), polyvinylidene chloride (PVDC), polyvinyl chloride (PVC) and acrylonitrile-butadiene-styrene (ABS). is selected from In one embodiment, the substrate includes the first material. In one embodiment, the first material is bonded, attached, coated, or sputtered onto the first surface. In one embodiment, the first material comprises a hydrophilic gasket. In one embodiment, the one or more regions are adapted to promote wetting by the first liquid by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents. do.

In one embodiment, the first surface includes one or more ridges surrounding one or more fluid ports. In one embodiment, the first surface includes one or more depressions surrounding one or more fluid ports. In one embodiment, the first surface is adapted to stably retain one or more aqueous droplets. In one embodiment, the first surface is adapted to stably retain one or more non-aqueous droplets. In one embodiment, the first surface is adapted to stably retain one or more oil droplets.

The present invention also contemplates a system comprising a device for retaining a droplet. In one embodiment, the system comprises: a) a first substrate comprising a first surface comprising a first set of one or more fluid ports, wherein the first surface comprises a first liquid in the first set of fluid ports; a first substrate adapted to stably retain one or more liquid droplets, b) a second substrate comprising a second surface comprising a second set of one or more fluid ports, and c) a first set of fluid ports. and a mechanism for fluidly contacting (connecting) the first set and the second set of fluid ports.

The present invention also contemplates a method of retaining a drop that can be combined with establishing a fluid connection. In one embodiment, a) a first substrate comprising a first surface comprising a first set of one or more fluid ports, wherein the first surface comprises a first liquid in the first set of fluid ports providing a first substrate adapted to stably retain at least one droplet, b) providing a second substrate comprising a second surface comprising a second set of one or more fluid ports, and c) ) A method of establishing a fluid connection is contemplated, comprising contacting (e.g., through controlled engagement) a first set of fluid ports and a second set of fluid ports. In a preferred embodiment, the contacting of step c) includes aligning the first set of fluid ports and the second set of fluid ports and contacting the aligned sets of ports.

In one embodiment, the present invention contemplates systems and methods for contacting a microfluidic device with a fluid source in a drop-to-drop connection. In one embodiment, the invention provides a device comprising: a) i) a fluid source comprising a first protruding fluid droplet and in fluid communication with a first fluid port located on the first abutment surface; and ii) providing a microfluidic device comprising a microchannel comprising a second protruding fluid droplet and in fluid communication with a second fluid port on the second abutment surface; and b) the first protruding fluid droplet and the second fluid droplet are connected together drop-to-point, such that fluid flows from the fluid source through the first fluid port to the second fluid port of the microfluidic device. Consider methods that include a flow step. In one embodiment, the invention provides a device comprising: a) a fluid source adapted to support a first protruding fluid droplet and in fluid communication with a first fluid port located on the first abutment surface; b) a microfluidic device adapted to support a second protruding fluid droplet and comprising a microchannel in fluid communication with a second fluid port on the second abutment surface; and c) the first protruding fluid droplet and the second fluid droplet are connected together drop-to-point, such that fluid flows from the fluid source through the first fluid port to the second fluid port of the microfluidic device. Consider a system that includes mechanisms that allow flow. In one embodiment, a first protruding fluid droplet protrudes downwardly from the first abutment surface and a second protruding fluid droplet protrudes upwardly from the second abutment surface. In one embodiment, a first protruding fluid droplet protrudes upwardly from the first abutment surface and a second protruding fluid droplet protrudes downwardly from the second abutment surface. In one embodiment, the mechanism elevates the second abutment surface into contact with the first abutment surface. In another embodiment, the mechanism elevates the first abutment surface into contact with the second abutment surface. In yet another embodiment, the mechanism lowers the second abutment surface into contact with the first abutment surface. In yet another embodiment, the mechanism lowers the first abutment surface into contact with the second abutment surface.

In one embodiment, the present invention contemplates that dripping is controlled by surface treatment. In one embodiment of the system, the first bonding surface includes an area surrounding the first fluid port, the area adapted to resist wetting by the fluid. In one embodiment the region is adapted to be hydrophobic. In one embodiment, the region includes a first material selected to resist wetting by the fluid. It is not intended that the invention be limited to the nature of the first material. However, in one embodiment, the first material is poly-tetrafluoroethylene (PTFE), perfluoroalkoxy alkane (PFA), fluorinated ethylenepropylene (FEP), polydimethylsiloxane (PDMS), nylon (some grades are hydrophobic), polypropylene, polystyrene, and polyimide. The invention is not intended to be limited by the nature of the first material adhering to the surface. However, in one embodiment, the first material is bonded, adhered, coated or sputtered onto the first bonding surface. The present invention also contemplates adding features having a surface that is inherently hydrophobic, or a surface that can be rendered hydrophobic. In one embodiment, the first material includes a hydrophobic gasket. The invention is not intended to be limited to the particular treatment method used to modify the surface or area of the surface. However, in one embodiment, said region of said first bonding surface is adapted to resist wetting by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents. .

Although embodiments of adapting a surface or a region of a surface to resist wetting have been discussed above, the present invention provides a method wherein the first bonding surface includes a region surrounding the first fluid port, wherein the region resists wetting by the fluid. Embodiments are contemplated that are adapted to facilitate. In one embodiment, the region is adapted to be hydrophilic. In one embodiment, the region comprises a first material selected to promote wetting by the first liquid. The present invention is not intended to be limited to a particular first material that promotes wetting. However, in one embodiment, the first material is polymethylmethacrylate (PMMA), polyvinyl alcohol (PVOH), polycarbonate (PC), polyether ether ketone (PEEK), polyethylene terephthalate (PET), Polyfulphone, polystyrene, polyvinyl acetate (PVA), nylon (some grades are hydrophilic), polyvinyl fluoride (PVF), polyvinylidene chloride (PVDC), polyvinyl chloride (PVC), and acrylonitrile-butadiene- It is selected from the group consisting of styrene (ABS). Additionally, the present invention is not intended to be limited by the technique by which the first material is attached to the surface. However, in one embodiment, the first material is bonded, adhered, coated or sputtered onto the first bonding surface. The present invention also contemplates introducing structures or features having surfaces that are inherently hydrophilic, or surfaces that can become hydrophilic. For example, in one embodiment, the first material includes a hydrophilic gasket. Additionally, the present invention is not intended to be limited to treatments that promote wetting. For example, in one embodiment, said region of said first bonding surface is adapted to promote wetting by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents. I get angry.

The invention also provides structural and geometrical features that can be molded or formed as part of a surface, attached to a source, deposited on, printed on or bonded to a source, or machined, etched or ablated into a surface. Consider wealth. For example, in one embodiment, the first abutment surface includes one or more ridges surrounding the first fluid port. In another embodiment, the first bonding surface includes one or more depressions surrounding the first fluid port.

The invention is also not limited to drop-to-drop connections with aqueous fluids only. In one embodiment, the first abutment surface is adapted to stably retain aqueous protrusion fluid droplets; however, in another embodiment, the first abutment surface is adapted to stably retain non-aqueous protrusion fluid droplets, such as, but not limited to, oil-based protrusion droplets. It is adapted to hold.

The present invention also contemplates a method of fusing drops using a drop-to-drop approach. In one embodiment, the invention provides a device comprising: a) i) a fluid source comprising a first protruding fluid droplet and in fluid communication with a first fluid port located on the first abutment surface; and ii) providing a microfluidic device or chip comprising a microchannel comprising a second protruding fluid droplet and in fluid communication with a second fluid port on the second bonding surface; and b) connecting the first protruding fluid droplet and the second fluid droplet together drop-to-point, thereby causing the first and second fluid droplets to fuse, such that fluid flows from the fluid source to the first fluid port. Consider a method of fusing droplets comprising flowing a fluid through a second fluid port of the microfluidic device. In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel. The present invention is not intended to be limited to any particular orientation or two bonding surfaces. In one embodiment, a first protruding fluid droplet protrudes downwardly from the first abutment surface and a second protruding fluid droplet protrudes upwardly from the second abutment surface. In another embodiment, a first protruding fluid droplet protrudes upwardly from the first abutment surface and a second protruding fluid droplet protrudes downwardly from the second abutment surface. The present invention is not intended to be limited to methods of bringing drops together. In one embodiment, step b) includes raising the second bonding surface upward into contact with the first bonding surface. In another embodiment, step b) includes raising the first bonding surface upward into contact with the second bonding surface. In yet another embodiment, step b) includes lowering the second bonding surface into contact with the first bonding surface. In yet another embodiment, step b) includes lowering the first bonding surface into contact with the second bonding surface. In a preferred embodiment, the drop-to-drop connection does not allow air to enter the fluid injection port.

The present invention contemplates surface treatments to promote wetting. In one embodiment, the first bonding surface includes an area surrounding the first fluid port, the area adapted to promote wetting by the fluid. In one embodiment, the region is adapted to be hydrophilic. In one embodiment, the region comprises a first material selected to promote wetting by the fluid. It is not intended to limit the invention to any particular first material, but in one embodiment, the first material is polymethylmethacrylate (PMMA), polyvinyl alcohol (PVOH), polycarbonate (PC), Polyether ether ketone (PEEK), polyethylene terephthalate (PET), polyfulfone, polystyrene, polyvinyl acetate (PVA), nylon, polyvinyl fluoride (PVF), polyvinylidene chloride (PVDC), polyvinyl chloride (PVC) ) and acrylonitrile-butadiene-styrene (ABS). While not intended to limit the invention to any particular attachment approach, in one embodiment, the first material is bonded, adhered, coated or sputtered onto the first bonding surface.

In some embodiments, the present invention contemplates adding features or structures to a surface, including structures having an inherently hydrophilic surface (or a surface that can become hydrophilic). In one embodiment, the first material comprises a hydrophilic gasket.

The present invention is not intended to be limited to any particular surface treatment technique. However, in one embodiment, said region of said first bonding surface is adapted to promote wetting by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents. .

Additional structures may be molded or otherwise formed within or on the surface. For example, in one embodiment, the first abutment surface includes one or more ridges surrounding the first fluid port. In another embodiment, the first bonding surface includes one or more depressions surrounding the first fluid port.

As mentioned above, the fluid need not be an aqueous fluid. In one embodiment the invention is adapted to allow the first abutment surface to stably retain an aqueous protruding fluid droplet, but in another embodiment the first abutment surface is adapted to stably retain a non-aqueous protrusion fluid droplet, e.g. It is contemplated that it is adapted to hold limited oily protruding droplets.

The present invention also contemplates a system for connecting ports together. In one embodiment, the system comprises a) a first substrate comprising a first fluid port, b) a second substrate comprising a second fluid port, c) adapted to align the first port and the second port. a guide mechanism, and (optionally) d) a retaining mechanism adapted to maintain contact between the first and second substrates. Although the invention is not intended to be limited to any particular guide mechanism, in one embodiment the guide mechanism is a guide track located on the first substrate, the guide track being configured to engage a portion of the second substrate. Although the invention contemplates embodiments where the retaining mechanism is on a first or second substrate, in one embodiment the retaining mechanism is a clip positioned on the second substrate, the clip being configured to engage the first substrate. .

In another embodiment, the invention provides a substrate comprising a) a first substrate comprising a first set of one or more fluid ports, b) a second substrate comprising a second set of one or more fluid ports, c) a first substrate of ports. Consider a system comprising: a guide mechanism adapted to align the first set and a second set of ports, and d) a retaining mechanism adapted to maintain contact of the first and second substrates. Again, various guiding mechanisms are contemplated (discussed herein). In one embodiment, the guide mechanism includes a guide shaft, or a hole, groove, orifice, or other cavity configured to receive the guide shaft. However, in one embodiment, the guide mechanism is a guide track located on the first substrate, and the guide track is configured to engage a portion of the second substrate. Again, various retention mechanisms are contemplated (discussed herein). However, in one embodiment, the retaining mechanism is a clip positioned on the second substrate, and the clip is configured to engage the first substrate.

The present invention also contemplates a method of connecting ports in a manner that establishes a fluid connection. In one embodiment, the invention provides a method comprising: a) providing a first substrate comprising a first fluid port, a second substrate comprising a second fluid port, and a guide mechanism adapted to guide the second substrate, b ) engaging the second substrate with the guide mechanism, c) aligning the first and second sets of fluid ports with the aid of the guide mechanism, and d) contacting the first and second fluid ports, thereby establishing a fluid connection. Consider a method of establishing a fluid connection, comprising establishing a. Although a variety of guide mechanisms are contemplated, in one embodiment the guide mechanism includes a guide track positioned on the first substrate, the guide track configured to engage a portion of the second substrate. In one embodiment of this method of establishing a fluidic connection, the second substrate comprises a microfluidic device comprising a bonding surface, and the second fluid port is located on the bonding surface and protrudes above the bonding surface. Includes. In a further embodiment, the first substrate includes a bonding surface, and the first fluid port is located on the bonding surface and includes a protruding droplet. Still further in this embodiment, the contacting of step d) results in a drop-to-drop connection when the first and second fluid ports establish a fluid connection. Preferably, the drop-to-drop connection does not allow air to enter one or more fluid injection ports. Although the invention is not limited to the manner of alignment, in one embodiment the alignment of step c) comprises sliding the second substrate by a guide track. Various designs and configurations for the guide track are contemplated, but in one embodiment the guide track includes first and second sections, the first section being configured to support the alignment of step c), and the second section is formed to support the contact of step d).

The present invention contemplates embodiments where the retaining mechanism is on the first substrate, but in one embodiment, the second substrate comprises a retaining mechanism adapted to maintain contact of the first and second substrates. In some embodiments, the retaining mechanism automatically engages when the first and second substrates contact to establish a fluid connection. However, in one embodiment, the invention includes an activating step e) activating the retention mechanism.

Although a two substrate system is described above, the present invention also contemplates a three substrate system. In one embodiment, the system comprises: a) a first substrate comprising a first fluid port, b) a second substrate comprising a second fluid port, c) a third substrate configured to support the second substrate; d) a guide mechanism adapted to align the first port with respect to the second port, and e) retaining mechanism means adapted to maintain contact of the first and second substrates.

As previously mentioned, a variety of guidance mechanisms are contemplated (discussed herein). In one embodiment, the guide mechanism includes a guide shaft, or a hole, groove, orifice, or other cavity configured to receive the guide shaft. There may be one or more guide shafts or other protrusions on one substrate, and one or more holes, grooves, orifices or other cavities on another substrate may be configured to receive one or more guide shafts or other protrusions. In one embodiment, the guiding mechanism includes a guiding track. The guide track(s) can have any orientation (eg, coming from the top rather than from one side). The present invention contemplates that the guide mechanism may be attached to a first, second or third substrate, but in one embodiment the guide track is located on the first substrate. Although the invention contemplates embodiments wherein the second or third substrate has features or structures configured to engage a guide mechanism, in one embodiment, the invention provides that the third substrate includes an edge configured to engage the guide track. Consider doing it. In one embodiment, the second substrate includes an edge configured to engage the guide track. The present invention contemplates embodiments where the holding mechanism is located on the first or second substrate; however, in one embodiment, the holding mechanism is located on the third substrate. As previously mentioned, a variety of retention mechanisms are contemplated. In one embodiment, the retention mechanism includes a clip configured to engage the first substrate. In another embodiment, the holding mechanism includes a clamp configured to engage the first substrate under conditions such that contact between the first and second substrates is maintained. In yet another embodiment, the retaining mechanism includes a stud configured to engage a hole on the first substrate. In yet another embodiment, the retaining mechanism frictionally engages a portion of the first substrate. In one embodiment, the retaining mechanism is selected from the group consisting of adhesives (eg, laminates), heat stakes, and screws.

Although the invention contemplates a system in which the components of the system are as described (see above), the invention also contemplates an assembly in which the components are arranged, attached or connected in a particular manner. In one embodiment, the invention provides a) a first substrate comprising a first fluid port and a guide mechanism and positioned in contact with and relative to a second substrate, b) a substrate comprising a second fluid port and supported by a carrier. Consider an assembly comprising: two substrates, c) a carrier including a portion engaging the guide mechanism of the first substrate, wherein the first and second ports are aligned to allow fluid communication. The present invention contemplates embodiments in which a retaining mechanism is positioned on the first or second substrate, but in one embodiment, the carrier further adds a retaining mechanism for maintaining the contact between the first and second substrates. Included as. A variety of guiding mechanisms are contemplated (and described herein), but in one embodiment, the guiding mechanism includes a guide track. The present invention is not limited to a single guide track, and two or more guide tracks may be used. For example, in one embodiment a guide track is located on one or more sides of the first substrate. In one embodiment, the carrier portion that engages the first substrate includes one or more edges configured to engage the guide track.

Although various retention mechanisms are contemplated (and described herein), in one embodiment of the assembly, the retention mechanism includes a clip configured to engage the first substrate. In another embodiment, the retaining mechanism includes a clamp configured to engage the first substrate. In yet another embodiment, the retaining mechanism includes a stud configured to engage a hole on the first substrate. In a particular embodiment, the retaining mechanism frictionally engages a portion of the first substrate. In one embodiment, the retaining mechanism is selected from the group consisting of adhesives (such as, but not limited to, laminates), heat stakes, and screws.

The present invention also contemplates a method of establishing a fluidic connection by bringing together fluidic ports comprising three substrates. In one embodiment, the invention provides a) a first substrate comprising a first fluid port, a second substrate comprising a second fluid port, a third substrate configured to support the second substrate, and a guide mechanism. step; b) aligning the first and second ports by the guide mechanism; and c) contacting the first port and the second port under conditions such that a fluid connection is established between the first and second substrates. In one embodiment of this three substrate method, the second substrate comprises a microfluidic device comprising a bonding surface, and the second fluid port is located on the bonding surface and includes a droplet protruding above the bonding surface. . Additionally in this embodiment, the first substrate includes a bonding surface, and the first fluid port is located on the bonding surface and includes a protruding droplet. Still further in this embodiment, the contacting of step c) results in a drop-to-drop connection when the first and second fluid ports establish a fluid connection. Preferably, the drop-to-drop connection does not allow air to enter one or more fluid injection ports.

Again, various guide mechanisms are considered and described herein. In one embodiment, the guiding mechanism includes a guiding track. The invention contemplates that the guide track is located on the first, second or third substrate, but in a preferred embodiment the guide track is located on the first substrate. In one embodiment, the third substrate includes an edge configured to engage the guide track. Although the invention is not intended to be limited to any particular technique for alignment, in one embodiment, the invention contemplates that the alignment of step b) comprises sliding the third substrate by the guide track. In one embodiment, the guide track comprises first and second sections, the first section being configured to support the alignment of step b) and the second section being configured to support the contacting of step c). . In one embodiment, the first section is linear and the second section is curved. In yet another embodiment, the guiding mechanism comprises a mechanism by which the third substrate rotates or pivots during step d). For example, in one embodiment, the guiding mechanism includes a hinge, joint, or pivot point.

Although the present invention contemplates embodiments wherein the retaining mechanism is positioned on the first or second substrate, in one embodiment, the present invention provides a retaining mechanism for maintaining the alignment of the first and second ports. Additionally includes. Again, a variety of retention mechanisms are contemplated. In one embodiment, the retention mechanism includes a clip configured to engage the first substrate. In one embodiment, the holding mechanism includes a clamp configured to engage the first substrate under conditions such that contact between the first and second substrates is maintained. In yet another embodiment, the retaining mechanism includes a stud configured to engage a hole on the first substrate. In yet another embodiment, the retaining mechanism frictionally engages a portion of the first substrate. In one embodiment, the retaining mechanism is selected from the group consisting of adhesives (such as, but not limited to, laminates), heat stakes, and screws. The invention also contemplates embodiments where the third substrate is a carrier for the second substrate.

In one embodiment, the invention provides a) a first substrate comprising a guide mechanism and a first fluid port on the first bonding surface, a second substrate comprising a bottom surface and a second fluid port on the second bonding surface, and a retaining surface. providing a carrier in contact with the lower surface of the second substrate and including one or more edges for engaging a mechanism and the guide mechanism; b) engaging the guide mechanism of the first substrate with one or more edges of the carrier; c) aligning the first and second ports by the guide mechanism; d) contacting the first bonding surface and the second bonding surface under conditions such that the first port contacts the second port to establish a fluid connection between the first and second substrates. , consider how to establish fluid connections. In one embodiment of this method, the second fluid port comprises a droplet that protrudes above the bonding surface of the second substrate. In one embodiment, the first fluid port includes a protruding droplet. In one embodiment, the contacting of step d) results in a drop-to-drop connection when the first and second fluid ports establish a fluid connection. Preferably, the drop-to-drop connection does not allow air to enter one or more fluid injection ports. A variety of guiding mechanisms are contemplated, but in one embodiment, the guiding mechanism includes a guiding track. The invention is not limited to embodiments with only one guide track; two or more guide tracks may be used. In one embodiment, guide tracks are located on one or more sides of the first substrate. In a preferred embodiment, the carrier comprises at least one edge configured to engage the guide track. Various alignment approaches are contemplated, but in one embodiment, the alignment of step c) includes sliding the carrier by the guide track. Various designs and configurations for the guide track are contemplated, but in one embodiment, the guide track includes first and second sections, the first section being configured to support the alignment of step c), and the second section The compartment is formed to support the contact of step d). In one embodiment, the first section is linear and the second section is curved. In yet another embodiment, the guiding mechanism comprises a mechanism by which the carrier rotates or pivots during step d). In this embodiment, the guiding mechanism may include a hinge, joint, socket or other pivot point.

In some embodiments, the retaining mechanism automatically engages when or after contact is made in step d). However, in one embodiment, the invention further comprises the step of e) activating the retention mechanism under conditions such that the alignment of the first and second ports is maintained. Again, a variety of retention mechanisms are contemplated. In one embodiment, the retention mechanism includes a clip configured to engage the first substrate. In one embodiment, the holding mechanism includes a clamp configured to engage the first substrate under conditions such that contact between the first and second substrates is maintained. In one embodiment, the retaining mechanism includes a stud configured to engage a hole on the first substrate. In one embodiment, the retaining mechanism frictionally engages a portion of the first substrate. In one embodiment, the retaining mechanism is selected from the group consisting of adhesives (such as, but not limited to, laminates), heat stakes, and screws.

The invention also contemplates devices for perfusing cells, such as devices that create a flow of fluid (e.g., culture medium) by applying pressure to a fluid reservoir. In one embodiment, the present invention contemplates an apparatus comprising an actuating assembly configured to move a pressure manifold comprising an integrated valve. In one embodiment, the device further comprises an elastomeric membrane. In one embodiment, the valve comprises a Schrader valve. In one embodiment, the pressure manifold includes an abutment surface having pressure points. In one embodiment, the device further includes a pressure controller. In one embodiment, the pressure controller is configured to apply pressure through the pressure point. In one embodiment, the actuation assembly includes a pneumatic cylinder operably connected to the pressure manifold. In one embodiment, the bonding surface further comprises an alignment feature configured to align the microfluidic device or chip when the microfluidic device or chip engages the bonding surface. In one embodiment, the device is a culture module for perfusing cells. In one embodiment, the microfluidic chip engages a perfusion manifold assembly (the alignment features are configured to align the perfusion manifold assembly). In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

The present invention also contemplates systems wherein the device for transmitting pressure is connected to a plurality of microfluidic devices, and more preferably, the plurality of microfluidic devices (e.g., the various embodiments of perfusion disposables discussed herein). are connected simultaneously (however, they can be connected individually or sequentially if desired). In one embodiment, the invention comprises a device comprising a) an actuating assembly configured to move a pressure manifold comprising an integrated valve, the pressure manifold b) a plurality of microfluidic devices (e.g., as disclosed herein). Consider a system that is in contact with (various embodiments of perfusion disposables discussed in). In one embodiment, the pressure manifold further comprises an elastomeric membrane, and the elastomeric membrane is in contact with the microfluidic device. In one embodiment, the microfluidic device is a perfusion disposable device. In one embodiment, the valve comprises a Schrader valve. In one embodiment, each microfluidic device is covered with a cover assembly comprising a cover having a plurality of ports, the pressure manifold comprising an abutment surface having pressure points corresponding to ports on the cover, and the pressure manifold A pressure point on the mating surface of the fold contacts the port of the cover assembly. In one embodiment, the port comprises a through-hole port that mates with corresponding holes in the strainer and gasket. In one embodiment, the device further includes a pressure controller. In one embodiment, the pressure controller is configured to apply pressure through the pressure point. In one embodiment, the actuation assembly includes a pneumatic cylinder operably connected to the pressure manifold. In one embodiment, the abutment surface of the pressure manifold further comprises an alignment feature configured to align the microfluidic device when the microfluidic device engages the abutment surface. In a preferred embodiment, the device is a culture module for perfusing cells. In one embodiment of this culture module, the culture module is configured to receive one or more trays, each tray comprising a plurality of microfluidic devices. In one embodiment, the culture module further includes a user interface for controlling the culture module. In one embodiment, each tray includes multiple perfusion manifold assemblies. In one embodiment, the microfluidic chip engages each perfusion manifold assembly (the alignment features of the pressure manifold mating surface are configured to align each perfusion manifold assembly). In one embodiment, the microfluidic chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device includes cells on a membrane and/or in or on a channel.

The present invention also provides for the use of culture modules to form cells (e.g., cells in the microchannels of a microfluidic device, such as the various embodiments of perfusion disposables discussed herein, with or without seeding guides of the type described herein). Consider a method of perfusing cells without first seeding them into the microfluidic device. In one embodiment, the invention provides A) a culture comprising: a) an actuating assembly configured to move a plurality of microfluidic devices relative to a pressure manifold, ii) a pressure manifold comprising a bonding surface having pressure points; module; and b) a plurality of microfluidic devices, each microfluidic device comprising: i) one or more microchannels containing living cells, ii) one or more reservoirs containing culture medium, and iii) a pressure manifold. comprising a cover assembly over the one or more reservoirs, the cover having ports corresponding to pressure points on the mating surfaces; B) installing the plurality of microfluidic devices on or in the culture module; and C) simultaneously (or sequentially) contacting the port on the cover of each microfluidic device of the plurality of microfluidic devices with the bonding surface of the pressure manifold, so that the culture medium flows from the reservoir to the microfluidic device. A method of perfusing a cell is contemplated, comprising the step of perfusing the cell by bringing the port into contact with the pressure point under conditions that allow flow into the microchannel of a device. In one embodiment, the plurality of microfluidic devices are positioned on one or more trays prior to step B), and the installation of step B) moves at least a subset of the plurality of microfluidic devices simultaneously to the culture module. It includes In one embodiment, the simultaneous contacting of step C) comprises moving the plurality of microfluidic devices upwardly relative to the abutment surface of the pressure manifold through an actuation assembly. In another embodiment, the present invention provides a culture module comprising: a) a) an actuation assembly configured to move a pressure manifold, ii) a pressure manifold comprising an abutment surface having pressure points; and b) a plurality of microfluidic devices, each microfluidic device comprising: i) one or more microchannels containing living (viable) cells, ii) one or more reservoirs containing culture medium, and iii) ) comprising a cover assembly over the one or more reservoirs, including a cover having ports corresponding to pressure points on a pressure manifold mating surface; B) installing the plurality of microfluidic devices on or in the culture module; and C) simultaneously (or sequentially) contacting the port on the cover of each microfluidic device of the plurality of microfluidic devices with the bonding surface of the pressure manifold, so that the culture medium flows from the reservoir to the microfluidic device. A method of perfusing a cell is contemplated, comprising the step of perfusing the cell by bringing the port into contact with the pressure point under conditions that allow flow into the microchannel of a device. In this embodiment, multiple microfluidic devices are connected simultaneously. They can then be simultaneously disconnected or disconnected from the pressure manifold. In one embodiment, the plurality of microfluidic devices are positioned on one or more trays (or nests) prior to step B), and the installation of step B) involves at least a subset (at least three) of the plurality of microfluidic devices. ) and simultaneously moving them to the culture module. In one embodiment, the simultaneous contact of step C) is achieved by moving the mating surface of the pressure manifold downwardly onto the cover assembly of the plurality of microfluidic devices through an actuation assembly. In one embodiment of the perfusion method, the microfluidic device includes a microfluidic chip (such as, but not limited to, the microfluidic chip shown in Figure 3A with one or more microchannels and ports) engaged with a perfusion manifold assembly, The assembly includes i) a cover or shroud configured to act as a top of one or more fluid reservoirs, ii) one or more fluid reservoirs, iii) a fluid backplane in fluid communication with and beneath the fluid reservoir(s), and iv) micro and a protruding member or skirt that engages (directly) a fluid chip or (indirectly) engages a carrier containing a microfluidic chip. Perfusion is preferably performed at a rate that provides (or maintains) viability of greater than 80%, and more preferably greater than 90%, and most preferably greater than 95%, of the cells contained within the microfluidic chip. In one embodiment, the assembly further includes a capping layer underneath the fluid reservoir(s). In one embodiment, the fluid backplane includes a resistor. In a preferred embodiment, the microfluidic chip environment remains sterile during the perfusion.

The present invention also provides for cells (e.g., cells in microchannels of a microfluidic device, such as the various embodiments of perfusion disposables discussed herein), wherein the cells are first seeded with or without a seeding guide of the type described herein. seeding the microfluidic device) to control the pressure during perfusion, e.g., in one embodiment, the pressure is reliably maintained at 1 pKa (+ or - 0.5 pKa, and more preferably + or - 0.15 pKa). Consider taking control. In one embodiment, the invention provides A) a) a plurality of microfluidic devices, b) one or more pressure actuators, each microfluidic device comprising: i) one or more microchannels containing living cells, ii ) comprising one or more reservoirs containing culture medium, B) coupling the pressure actuator to at least one of the reservoirs, wherein the coupling is such that the actuated pressure causes perfusion of at least a portion of the living cells. C) switching on said one or more pressure actuators between two or more pressure setpoints, thereby controlling the pressure during perfusion of said cells. Consider ways to control it. In another embodiment, the present invention provides a device comprising: A) a) an actuating assembly configured to move an i) a pressure manifold, ii) a pressure manifold comprising an abutment surface having a pressure point, and iii) applying pressure to the pressure point. a culture module including one or more pressure controllers for providing; and b) a plurality of microfluidic devices, each microfluidic device comprising: i) one or more microchannels containing living cells, ii) one or more reservoirs containing culture medium, and iii) a pressure manifold. comprising a cover assembly over the one or more reservoirs, the cover having ports corresponding to pressure points on the mating surfaces; B) installing the plurality of microfluidic devices on or in the culture module; C) Simultaneously contacting the port on the cover of each microfluidic device of the plurality of microfluidic devices with the bonding surface of the pressure manifold, so that culture medium flows from the reservoir into the microchannel of the microfluidic device. perfusing the cells by bringing the port into contact with the pressure point under flow conditions; and D) turning the one or more pressure controllers off for a predetermined period and on (or switching them on and off) for a predetermined period (or switching them on and off between two or more settings) to control pressure while perfusing the cells. Consider a method of controlling pressure while perfusing cells, comprising: In one embodiment, the switching is between a setpoint of 1 kPa and 0.5 kPa, with good conversion within that range. In one embodiment, the switching has three levels: 2 kPa, 1 kPa and 0 kPa in some advanced methods. In one embodiment, the pressure controller is turned on and off (or between set points) in a switching pattern (e.g., turns them off and on, or turns them off and on repeatedly at defined intervals between set points). In a preferred embodiment, the switching pattern is selected such that the average value of the liquid operating pressure in said one or more reservoirs corresponds to the desired value. For cells, the desired values are typically low. For example, in one embodiment, the switching pattern is selected to maintain the average gas pressure below 1 kPa. In one embodiment of the perfusion and pressure control method, the microfluidic device includes a microfluidic chip (such as, but not limited to, the microfluidic chip shown in FIG. 3A having one or more microchannels and ports) that engages a perfusion manifold assembly. wherein the assembly comprises i) a cover or shroud configured to act as a top of one or more fluid reservoirs, ii) one or more fluid reservoirs, iii) a fluid backplane under and in fluid communication with said fluid reservoir(s), and iv) a protruding member or skirt which engages (directly) with the microfluidic chip or (via which) it engages indirectly with the carrier containing the microfluidic chip. Perfusion is preferably performed at a rate that provides viability of greater than 80%, and more preferably greater than 90%, and most preferably greater than 95% of the cells contained within the microfluidic chip. In one embodiment, the assembly further includes a capping layer underneath the fluid reservoir(s). In one embodiment, the fluid backplane includes a resistor. In one embodiment, the port on the cover or lid is associated with a strainer. In one embodiment, the filter is a 0.2 micrometer, 0.4 micrometer, or 25 micrometer filter. In a preferred embodiment, the microfluidic chip environment remains sterile during the perfusion. In one embodiment, cycling the pressure regulator on and off brings the average value of the pressure close to the desired value, but confirmed by a microfluidic device or chip by introducing a resistive filter into the inlet portion of the cover of the perfusion manifold assembly. The resulting maximum and minimum values are much closer to the desired values.

A pressure shroud is contemplated as a device within or otherwise associated with a microfluidic device that allows pressurization of one or more fluid sources (e.g., reservoirs). In one embodiment, the present invention contemplates a pressure shroud comprising a plurality of ports configured to engage a pressure manifold. In one embodiment, the port is associated with a strainer. In one embodiment, the cover is associated with a gasket. In one embodiment, the pressure cover is movable or removably attached to the microfluidic device, allowing improved access to elements (e.g., reservoirs) from within. In one embodiment, the present invention provides a method comprising: a) providing a pressure shroud comprising a plurality of ports configured to engage a pressure manifold, a microfluidic device comprising a fluid source, and a pressure manifold; b) positioning the pressure shroud over the fluid source, creating a positioned pressure shroud; and c) engaging the positioned pressure shroud with the pressure manifold under conditions where pressure is applied through the port to cause fluid from the fluid source to move into or through the microfluidic device. Consider. In one embodiment, the method further comprises d) disconnecting the positioned pressure shroud from the pressure manifold. The pressure cover can then (optionally) be removed and the microfluidic device can be used without the cover.

The invention also includes a) equipment for interfacing with a microfluidic device, b) a pressure cover comprising i) one or more fluid reservoirs and ii) one or more equipment-access ports and one or more reservoir-connection ports. A system comprising a microfluidic device in fluid communication therewith, wherein the pressure shroud is adapted to transfer pressure between at least one of the device-facing ports and at least one of the reservoir-facing ports. . In one embodiment, the equipment includes a pressure manifold (moving or non-moving). In one embodiment, one or more fluid reservoirs are disposed in a cartridge, the cartridge being in fluid communication with the microfluidic device. In one embodiment, one or more fluid reservoirs are disposed in the microfluidic device.

The present invention also contemplates as a device a pressure shroud comprising one or more equipment-facing ports and one or more reservoir-facing ports, wherein the pressure shroud comprises at least one of the equipment-facing ports and at least one of the reservoir-facing ports. and the pressure shroud is adapted to form a pressure connection with at least one fluid reservoir.

The present invention also contemplates as a device a pressure shroud comprising one or more channels, each channel comprising an equipment-connected end and a reservoir-connected end, the channels configured to transfer pressure between the equipment and the fluid reservoir. do.

1A shows a cover (or cover assembly) of a reservoir (the reservoir body may be made of acrylic, for example), a reservoir positioned over a backplane, a backplane in fluid communication with the reservoir, and one or more injection, discharge and (optionally) a vacuum port, and a skirt with side tracks for engaging a representative microfluidic device or “chip” (which may be made of plastic, such as PDMS), having one or more microchannels, a chip carrier (e.g. , which may be fabricated from a thermoplastic polymer such as acrylonitrile butadiene styrene (ABS), is shown next to (but not within) one embodiment of a chip, configured to support and retain the chip, e.g., where the chip is placed within a cavity. An exploded view of one embodiment of a flow-through manifold assembly, showing a carrier with dimensions to fit. 1B is the same embodiment of a perfusion manifold assembly, with a cover on and over a reservoir, and a chip inside a chip carrier fully connected to the skirt of the perfusion manifold assembly and in fluid communication with the reservoir. In one embodiment, each chip has two inlets, two outlets and (optionally) two connections for vacuum stretching. In one embodiment, putting the chips in fluid communication connects all six in one motion rather than connecting them one at a time. 1C is an exploded view of one embodiment of a flow manifold assembly, including a reservoir fluidically sealed by a capping layer and located above a fluid backplane (including a fluid resistor) located above the skirt (before the components are assembled). ), and each piece has dimensions that fit with respect to each other. In one embodiment, the skirt has a structure (e.g., made of polymer) that abuts or defines two open spaces, one of which is configured to receive a carrier with a chip therein. In one embodiment, the skirt has a structure that completely surrounds two “arms” extending outwardly to define one open space and a second open space for receiving the carrier. In one embodiment, the two arms have side tracks that slidably engage the carrier edge.
2A is an exploded view of one embodiment of a pressure cover or cover assembly including a pressure cover. In the depicted embodiment, the pressure shroud includes a plurality of ports (e.g., through-hole ports) that mate with corresponding holes in the strainer and gasket. The illustrated design of the holes in the gasket is intended to assist the gasket in maintaining the illustrated strainer in place. In alternative embodiments, the gasket opening may utilize a different shape than the opening in the cover. For example, a gasket can be molded to follow the contour of one or more reservoirs that are intended to form a fluid or pressure seal. In some embodiments, multiple gaskets may be used. In some embodiments, the filters and/or gaskets are secured using adhesives, heat stakes, bonding (ultrasonic, solvent-assisted, laser welding), clamped, or captured in the absence of a cover and/or additional substrate. It can be. Although the pressure shroud shown includes a through-hole port, alternative embodiments include one or more channels passing from at least one upper surface port to one or more lower surface ports, wherein the lower surface port is an upper surface port. It doesn't have to be right below it. Figure 2B shows the same embodiment as the cover assembly shown in Figure 2A, with a strainer and gasket inside (and underneath) the cover. Figure 2C-1 is a cross-sectional view of one embodiment of a cover assembly, showing a ridge or sealing tooth surrounding both through-hole ports in the cover. Figure 2C-2 is an enlarged view of a portion (circled) of Figure 2C-1. In the depicted embodiment, the cross-sectional shape of the sealing tooth is trapezoidal, although other contemplated embodiments utilize other tooth shapes, such as, but not limited to, semicircular, rectangular, polygonal, and triangular shapes. 2D is a top view of one embodiment of a reservoir chamber-cover assembly seal, showing the sealing tooth, vacuum chamber, and injection and discharge chambers. 2E-1 is a cross-sectional view of one embodiment of a cover assembly seal associated with a reservoir, showing a cover gasket and sealing tooth. Figure 2E-2 is an enlarged view of a portion (circled) of Figure 2E-1. As the pressure manifold (discussed below) engages the cover assembly, pressure forces the cover assembly (including the cover gasket) into the sealing teeth, forming a seal between the respective reservoir chambers.
Figure 3A shows one embodiment of a microfluidic device or chip, showing two channels with injection and discharge ports, as well as an (optional) vacuum port. 3B is a top schematic diagram of an alternative embodiment of a perfusion disposable or “pod” characterized by a transparent (or translucent) cover over a reservoir with a chip inserted therein. The chip can be seeded with cells and then placed in a carrier for insertion into perfusion disposables. FIG. 3C is a schematic diagram of an assembled perfusion disposable embodiment identical to that shown in FIG. 3B except that ports and cutouts on the cover assembly (above the chip inserted for visualization, imaging, etc.) are shown. Figure 3D is a schematic diagram of the same irrigation disposable embodiment of Figure 3C, but unassembled to illustrate the relationships of the cover, reservoir, skirt, chip and carrier.
FIG. 4A shows a side view of one embodiment of a chip carrier (with a chip inside) approaching a side track of a skirt of one embodiment of a flow-through manifold assembly, wherein the carrier is aligned with one of the side tracks. Aligned at an angle matching the angled front end portion, the carrier includes a retaining mechanism configured as an upwardly projecting clip. Without wishing to be bound by theory, a suitably large angle allows chip engagement without smearing or premature engagement of droplets present on the chip and/or perfusion manifold assembly during the insertion and alignment process. Figure 4B shows a side view of one embodiment of a chip carrier (with a chip inside) engaged with the side tracks of a skirt of one embodiment of a flow-through manifold assembly (not yet connected to the assembly). 4C shows a side view of one embodiment of a chip carrier (with a chip inside) fully engaged with the side tracks of a skirt of one embodiment of a flow-through manifold assembly (not yet connected to the assembly) (arrows indicate Shows the direction of movement required for a snap fit, thereby engaging the retaining mechanism to prevent movement). FIG. 4D shows a side view of one embodiment of a chip carrier (with a chip therein) removably connected to a perfusion manifold assembly, where the retaining mechanism is engaged to prevent movement. Although detachability and optionally reattachability are desirable in certain applications (e.g., to allow chip removal to allow cell addition, imaging, performing various assays), in alternative embodiments, the connections are not detachable. . For example, adhesive layers, glues and/or heat stakes can be used to provide a sturdy connection that may make detachment or reattachment difficult. 4E shows a perfusion manifold comprising 1) a sliding motion (4e-1), 2) a pivot (4e-2), and 3) a snap fit (4e-3) to provide alignment and fluidic connection in a single motion. A sliding overview schematically depicting one embodiment of the connection approach. 1) In the sliding step, the chip (or other microfluidic device) is inserted into the sliding carrier to align the fluid ports. 2) In the pivot step, the chip (or other microfluidic device) is pivoted until the port is in contact with the fluid. 3) At the clip or snap fit stage, provide the necessary force to provide a positive seal.
Figure 5 is a schematic diagram of one embodiment of the workflow (arrows depict each proceeding step), wherein the chip is connected (e.g., snapped) to a disposable perfusion manifold assembly ("perfusion disposable"). , the assembly is placed again on another assembly on the culture module, and the culture module is installed in the incubator. In alternative embodiments, the culture module may include features of an incubator (e.g., a source of heat and/or a source of warm, humid air) to eliminate the need for a separate incubator. The present invention contemplates a “disposable” embodiment, but the members are (alternatively) reusable (e.g., for cost considerations). In further embodiments of the workflow or method, a chip can be installed in a carrier, the carrier can be installed in a seeding guide (discussed and shown below), cells can be seeded on the chip, and the carrier can be removed from the seeding guide. can be removed and the carrier engaged with the irrigation disposable (remaining the same as the workflow shown in Figure 5).
Figure 6 shows one embodiment of a removable tray with multiple assemblies (with connected chips) positioned thereon, pressure on the bonding surfaces corresponding to ports on the covers of each perfusion manifold assembly held in the tray next to it. Shown is one embodiment of a culture module having points so that they can be placed together by a tray mechanism so that pressure can be applied through a pressure controller. This allows the tray mechanism to simultaneously attach and connect all flow manifold assemblies to pressure or flow controllers (whether raising the tray up or down to meet the tray) in a single motion.
Figure 7 is a side schematic diagram of another embodiment of a culture module, showing a platform for positioning a removable tray that moves up to the bonding surface so that pressure can be applied through a pressure controller (not shown).
Figure 8A is a schematic diagram of another embodiment showing a tray (or rack) and sub-tray (or nest) for transporting and inserting perfusion disposables (PD) into a pressure module with user connections on the outside of the housing. Figure 8B is a schematic diagram of another embodiment showing a tray (or rack) inserted within the housing of a culture module with user connections. The nested design, in which a tray is shown (as an example of the invention) holding multiple removable sub-trays, provides the user with the flexibility to remove or retain varying numbers of PDs depending on the application. For example, the user can transfer all trays to a biosafety cabinet to refill media or collect samples from all PDs in a tray, or control the remaining PDs in terms of temperature and gas content to image three PDs. A sub-tray of three PDs can be moved to the microscope stage without interruption, or a single PD can be removed or loaded for careful inspection or replacement.
9A shows a tray (or rack), sub-tray (or nest), and perfusion disposables (PD) positioned below a pressure manifold (but not engaged with it, so with sufficient clearance to remove them), and the actuating assembly (atmospheric pressure) Schematic diagram of the interior (open position) of one embodiment of a pressure module, including a pressure cylinder) on a pressure manifold. Although three microfluidic devices or perfusion disposables are shown, typically more are used at a time (e.g., 6, 9, or 12).
9B shows a tray (or rack), sub-tray (or nest), and perfusion disposables (PD) positioned below (in engagement with) a pressure manifold and an actuation assembly (including pneumatic cylinder) positioned above the pressure manifold. Schematic diagram of the interior of one embodiment of a pressure module (closed position). Again, three microfluidic devices or perfusion disposables are shown, but typically more are used at a time (eg, 6, 9 or 12).
10A is a schematic diagram of one embodiment of a pressure manifold 50, showing a PD engagement surface 54 with several PD engagement positions (in this case six engagement positions). 10B is a close-up of the engagement surface 54 of the pressure manifold 50, highlighting the spring shuttle 55, valve seal 56, and alignment feature 57 (which allows the PD to align with the manifold). Show the part that has been done. 10C shows the PD engagement surface 54 along an enlarged portion highlighting the shroud compressor 58, valve seal 56, and alignment feature 57 (allowing the PD to align with the manifold). A schematic diagram of another embodiment of a pressure manifold 50. FIG. 10D is a side schematic diagram of one embodiment of a pressure manifold 50, showing the PD engagement surface 54. 10E is a schematic diagram of one embodiment of a pressure manifold 50, showing the opposing surface 67. 10F shows one spring carrier (70) and spring (71) (among many) removed from the manifold body, one seal (72) (among many), shuttle (73) and valve body. One embodiment of a pressure manifold (50) showing the PD engagement surface (54) with the PD guide (68) and lower backer plate (69) removed, highlighted by showing (74) removed from the manifold body. This is a schematic diagram. The external spring 75 adapted to press the pressure manifold against the perfusion disposable is also highlighted by showing it removed. 10G is a schematic diagram of one embodiment of a pressure manifold 50, showing the opposing surface 67 (not the PD engagement surface) with the upper backer plate 76 and capping strip 77 removed. A trans-manifold channel 78 is shown adapted to derive and optionally distribute pressure and/or fluid from one or more pressure ports. Additionally, one screw 79 (out of several) and one gas port 80 (out of five, including both gas and vacuum ports) are shown by showing them removed from the manifold body 50. 10H is a schematic diagram of one embodiment of a pressure manifold 50, showing a top view of the manifold via channel 78 and one of several ports 81.
11A shows one embodiment of a valve 59 (Schrader valve) in a pressure manifold 50, showing the silicone membrane 60, shuttle 61, air inlet 62 and cover plate 63. This is a schematic diagram. FIG. 11B is a side view photograph of one embodiment of a valve for a pressure manifold, showing the valve seat 64 and membrane 60 acting as a valve seal, and FIG. 11C is a top view photograph. 11D is a schematic illustration of an interior side view of one embodiment of a pressure manifold 50, showing a plurality of valves 59, poppets 65, valve seals 66, and PD covers 11 in the manifold body. am. In operation (to engage the PD), the valve seal 66 is deflected by the displacement of the poppet 65.
12A shows one embodiment of a connection scheme comprising a tubing connection manifold 82 allowing four culture modules 30 (three shown) to be connected inside a single incubator 31 using one or more hub modules. Schematic diagram of the embodiment (the two circles provide an enlarged view of the first end 83 and the second end 84 of the connection). FIG. 12B is a photograph of the gas hub and vacuum hub (collectively 85) along tubing 86 for the connection shown in FIG. 12A.
Figure 13 is a photograph of one embodiment of an incubator (sealed from the outside by an external door) containing a shelf (not shown) capable of supporting a perfusion manifold assembly of the present invention. The incubator may have automated liquid handling, imaging, and sensing features for automated experiments, cell viability assessment, and/or collection of experiment results. In one embodiment, the microfluidic device is connected during incubation.
Figure 14 is a schematic diagram of one embodiment of connecting two microfluidic devices to introduce air bubbles into a microchannel. Figure 14A shows two fluidically primed devices with ports and microchannels not yet connected (fluids shown as meniscus). FIG. 14B shows the device of FIG. 14A contacted in such a way as to introduce an air bubble (air is shown midway between each meniscus) into the port (ultimately a microchannel).
Figure 15 is a schematic diagram of one embodiment of connecting two microfluidic devices (or a microfluidic device to a fluid source) using a drop-to-drop approach without creating air bubbles. Figure 15A shows two fluidically primed devices with microchannels with raised droplets formed on the surface of the device, more specifically directly on and above the ports, rather than in the fluid path or area around the ports. Figure 15B shows that drop surfaces typically join without introducing any air bubbles when the surfaces are close to each other during joining.
Figure 16A shows one embodiment of contacting a microfluidic device with a fluid source or another microfluidic device, where the microfluidic device is approached from the side. FIG. 16B shows one embodiment of contacting a microfluidic device with a fluid source or another microfluidic device, wherein the microfluidic device approaches from below the side, creating a drop-to-drop connection establishing fluid communication ( FIG. 16C ). It shows. Figure 16D shows another approach for contacting a microfluidic device with a fluid source or another microfluidic device, where the microfluidic device pivots.
17 is a schematic diagram showing a defined droplet 22 on the surface 21 of a microfluidic device 16 in a pathway or port.
18 is a schematic diagram showing a defined droplet 22 on the surface 21 of a microfluidic device 16 in the region of a path or port, where the droplet is on a molded pedestal or mount 42 and in the region of the port. It covers the inlet and protrudes above the port, which is in fluid communication with the microchannel.
19 is a schematic diagram showing a defined droplet 22 on the surface 21 of the microfluidic device 16 in the region of a passage or port, where the droplet is on a gasket 43 and covers the inlet of the port. , protrudes above the port, and the port is in fluid communication with the microchannel.
FIG. 20 is a schematic diagram showing a defined droplet 22 in which a portion of the droplet in the region of the path or port is located below the surface 21 of the microfluidic device 16, where the droplet is a shaped depression or depression ( 44) and covers the entrance of the port, a portion of which protrudes above the surface, and the port is in fluid communication with the microchannel.
FIG. 21 is a schematic diagram showing a defined droplet 22 with a portion of the droplet located below the surface 21 of the microfluidic device 16 in the region of the path or port, where the droplet is within the surrounding gasket and at the inlet of the port. covers, and a portion protrudes above the gasket.
Figure 22 is a schematic diagram showing a surface modification embodiment. Figure 22A utilizes a hydrophilic adhesive layer or sticker 45, with droplets 22 spreading from the edge of the sticker and bound by the surrounding hydrophobic surface. Figure 22b shows droplets 22 spread out on the hydrophilic surface of the device and confined by the surrounding hydrophobic surface.
Figure 23 is a schematic diagram of a surface modification embodiment (indicated by the arrow pointing "‡ below) utilizing surface treatment with mask 41.
Figure 24 is a schematic diagram of one embodiment of a drop-to-drop connection scheme that uses a combination of geometry and surface treatment to control droplets. FIG. 24A shows an embodiment of a microfluidic device or “chip” comprising fluidic channels and ports, with a raised area (e.g., a pedestal or gasket) at each port. Figure 24B shows a fluid-filled hydrophilic channel with the drop radius balanced at each end (i.e., at the port opening). 24C shows an upwardly protruding droplet 22 approaching (but not yet touching) a portion of the mating surface of a flow-through manifold assembly with a protruding droplet (in this case, droplet 23 protruding downwards). A portion of the microfluidic device of FIG. 24B is shown with . FIG. 24D shows the same portion of the microfluidic device of FIG. 24C with the upwardly protruding droplet 22 of the microfluidic device in contact (fused) with the downwardly protruding droplet 23 of the perfusion manifold assembly.
Figure 25 shows an embodiment of drop-to-drop connected using only surface treatment (i.e., without geometry, such as a pedestal or gasket). Figure 25A shows an embodiment of a perfusion manifold assembly including fluid channels and ports. Figure 25B shows a hydrophilic channel filled with fluid to a given level (e.g., to the height of the fluid column).
Figure 26 is a chart showing (without being bound by theory) the relationship between port diameter (in millimeters) and the maximum hydrostatic head that a stabilized drop can support (in millimeters).
27 shows an embodiment in which a microfluidic device (“chip”) is connected from below to a flow-through manifold assembly (top) at a port with an exhaust gasket 43, where the assembly does not cover or seal the gasket. , allowing any air trapped during connection to be vented (right arrow). It may be desirable to ensure that any air flows preferentially through the exhaust gasket rather than continuing to flow through the channel. In some embodiments, this preferential flow may be promoted by applying a first pressure (P1) to the fluid in the fluid channels of the assembly (left arrow) and a second pressure (P2) to the fluid in the microfluidic device channels. can be, where P1 and P2 are higher than the back pressure of the exhaust gasket. In some embodiments, pressure P1 and/or P2 is applied using a pressure source and/or gravity head. In some embodiments, pressure P1 and/or P2 is generated by flow resistance of the fluid.
28 shows another embodiment in which a microfluidic device 16 (“chip”) is connected from below to a perfusion manifold assembly 10 (top) at a port with an exhaust gasket 43, wherein the assembly covers the gasket (i.e., the gasket is surrounded by the assembly mating surface), but has a passageway in the assembly above the gasket to allow any air trapped during the connection to be vented.
29 shows another embodiment in which a microfluidic device 16 (“chip”) is connected from below to a perfusion manifold assembly 10 (top) at a port with an exhaust gasket 43, wherein the fluid The passage passes over the gasket (the gasket can be larger if desired). This embodiment facilitates the removal of trapped air, such as smaller bubbles, during connection, and without being bound by theory, because this allows the smaller bubbles to interact (“wet”) with the exhaust gasket.
30 and 31 show a flow path along an essentially linear rail or guide track of a fluid source, or microfluidic device, such as a perfusion manifold assembly (i.e., x A series of still life photos from the video showing one embodiment of a microfluidic device (with a droplet protruding from a gasket) moving (compare FIGS. 30A and 30B) along the x-axis in the /y-plane, where and a combination of z-axis movements (i.e., lateral and upward movements) fuse the droplets and connect the chips (Figure 31b).
32A shows one embodiment of a fluid backplane comprising a tortuous fluid resistor channel (91), a vacuum channel (92), and an exhaust channel (93). Figure 32b is an edge diagram. FIG. 32C shows a chip engagement relief 94 of the fluid backplane, acting as a fluid discharge port, along with assembly alignment features 95 and visualization cutouts 96 that allow for microscopy and other imaging.
Figure 33 shows a schematic diagram of an exemplary microfluidic device or “organ on a chip” device. The assembled device is schematically shown in Figure 33A. Figure 33b shows an exploded view of the device of Figure 33a.
Figure 34 is a schematic diagram showing an embodiment with two membranes.
35A shows first and second end caps 106 and 107 and first and second side panels 108 and 109 as components of one embodiment of an unassembled culture stand or holder 100. . Figure 35b shows the chip 16 and carrier 17 within the seeding guide, with the seeding guide approaching (but not engaged) the stand 100. Figure 35c shows six seeding guides containing carriers 17 (with chips) mounted on stand 100.
Figures 36A-C are photographs of an embodiment of a perfusion manifold assembly lacking a skirt (or other protrusion) with side tracks for engaging a chip (or other microfluidic device) in a carrier. Instead, the base 110 of the assembly 10 is configured to receive the carrier 17 from below (instead of from the side) with a Lego™ block type connection, i.e. the base 110 supports the carrier 17 While the handle or tab 18 of the carrier is configured to fit into the opening 112, it has a cavity 111 and an opening 112 dimensioned to receive the . Figure 36A is a top side view of assembly 10 before carrier 17 and chip 16 are engaged. 36B is a bottom side view of assembly 10 with fluid outlet port 94 configured to align with port 2 on chip 16. 36C shows the assembly 10 engaged with the carrier such that the carrier tab 18 is positioned in the opening 112.

Justice

“Bound number” is the dimensionless ratio of gravitational force to capillary force on the liquid interface. The higher the binding number of air, the more likely it is that the liquid interface is formed by gravity. The smaller the number of bonds, the more likely these surfaces are to form by capillary forces.

A “hydrophobic reagent” can be used to create a “hydrophobic coating” on a surface (or part thereof), such as a protrusion, platform or pedestal at or near a port, as well as a bonding surface (or part thereof). The present invention is not intended to be limited to particular hydrophobic reagents. In one embodiment, the present invention contemplates using silanes to make hydrophobic coatings, including but not limited to halogenated silanes and alkylsilanes. In this regard, the present invention is not intended to be limited to particular silanes; The choice of silane is limited only from a functional point of view, i.e. it renders the surface hydrophobic. The present invention also contemplates the use of commercially available products, such as Rain-X™ products (synthetic hydrophobic surface-applied products that bead water, most commonly used on glass automotive surfaces).

A surface or region on a surface is “hydrophobic” if its (e.g., advancing) contact angle to water is greater than approximately 90° (in many cases, it is desirable for both advancing and receding contact angles to be greater than approximately 90°). In one embodiment, the hydrophobic surface of the invention exhibits an advancing contact angle for water of approximately 90° to approximately 110°. In another embodiment, the hydrophobic surface has regions that exhibit an advancing contact angle for water greater than approximately 110°. In another embodiment, the hydrophobic surface has a region that exhibits a receding contact angle for water greater than approximately 100°. It is important to note that some liquids, especially some biological liquids, may contain ingredients that can coat the surface after being wetted with it, affecting its hydrophobicity. In the context of the present invention, it is provided that the surface resists such coating from the intended use liquid and that such coating does not produce advancing and/or receding contact angles of less than 90°, for example while the surface remains wetted by said liquid. may be important.

A surface or region on a surface is “hydrophilic” if its (e.g. advancing) contact angle to water is less than approximately 90°, more commonly less than approximately 70° (in many cases, both advancing and receding contact angles are approximately 90°). or preferably less than approximately 70°).

The measured contact angle can be within a certain range, i.e. between the so-called advancing (maximum) contact angle and the receding (minimum) contact angle. Equilibrium contact is within these values and can be calculated from them.

Hydrophobic surfaces “resist wetting” by aqueous liquids. If the contact angle formed by a first liquid on a material is greater than 90°, the material is said to resist wetting by the first liquid. The surface can resist wetting by aqueous and non-aqueous liquids, such as oils and fluorinated liquids. Some surfaces can resist wetting by both aqueous and non-aqueous liquids. Hydrophobic behavior is generally observed for surfaces with a critical surface tension of less than 35 dyne/cm. First, the decrease in critical surface tension is associated with oleophilic behavior, i.e. wetting of the surface by hydrocarbon oil. As the critical surface tension decreases below 20 dyne/cm, the surface resists wetting by hydrocarbon oils and is considered hydrophobic as well as oleophobic.

Hydrophilic surfaces “promote wetting” by aqueous liquids. If the contact angle formed by the first liquid on the material is less than 90°, more often less than 70°, the material is said to promote wetting by the first liquid.

As used herein, the phrases “connected,” “connected to,” “coupled to,” “in contact with,” and “in communication with” refer to any form of interaction between two or more entities, such as mechanical, electrical, etc. , refers to magnetic, electromagnetic, fluidic and thermal interactions. For example, in one embodiment, channels in a microfluidic device are in fluid communication with cells and (optionally) a fluid reservoir. Two components can be coupled to each other even though they are not in direct contact with each other. For example, two components can be coupled to each other through an intermediate component (eg, tubing or other conduit).

A “channel” is a path (straight, curved, single, multiple, network, etc.) through a medium (e.g., silicon, plastic, etc.) that allows the movement of liquids and gases. Accordingly, channels can connect different components, i.e., keep the components “in communication”, more particularly in “fluid communication”, and even more specifically in “liquid communication”. These components include, but are not limited to, liquid-inlet ports and gas outlets.

“Microchannels” are channels with dimensions of less than 1 millimeter and greater than 1 micrometer. Additionally, as used herein, the term “microfluidic” refers to a component that confines or directs fluid movement through one or more channels where one or more dimensions are 1 mm or less (microscale). A microfluidic channel may be larger than microscale in one or more directions, but the channel(s) will be microscale in at least one direction. In some examples, the geometry of a microfluidic channel can be configured to control the fluid flow rate through the channel (eg, increase channel height to reduce shear). Microfluidic channels can be formed in a variety of geometric shapes to facilitate a wide range of flow velocities through the channels.

The present invention relates to a variety of "microfluidic devices", including but not limited to microfluidic chips (e.g., shown in Figure 3A), perfusion manifold assemblies (without chip), and perfusion manifold assemblies engaged with microfluidic chips (e.g., (shown in Figure 3b). However, the methods described herein for engaging and perfusing microfluidic devices (e.g., by drop-to-drop connections) are not limited to the particular embodiments of the microfluidic devices described herein, but rather are general It can be applied to microfluidic devices, for example, devices having one or more microchannels and ports.

A “stable instillation” is one that does not experience significant movement from its intended location (e.g. remains in contact with a fluid port), preferably in a few seconds, more preferably in a minute, even more preferably in several minutes ( A drop of medium that does not experience a significant (>10%) change in volume or batch on the microfluidic device over a period of time (2-10 minutes). In a preferred embodiment, the present invention contemplates a stable drop during drop-to-drop engagement. A surface may inherently (e.g., because of what it is made of) be capable of holding the droplet stable, or it may be made to hold the droplet stable, meaning that the droplet will not spontaneously expand or move beyond a confined (or designated) area. . Stable drops do not experience significant changes in volume or placement. The present invention contemplates such spatial control of the drop, i.e. maintaining the drop within a defined spatial extent and/or maintaining the drop within a spatial extent of one or more regions. In a preferred embodiment, the present invention prevents the droplet from extending too far and ensures that the droplet is centered in the port (i.e., ensuring that the area immediately above the fluid port remains covered by the droplet). consider that In terms of preventing the droplet from expanding or spreading too far, the present invention in one embodiment contemplates keeping the droplet within the spatial extent of one or more areas. In a particularly preferred embodiment, the present invention contemplates preventing the droplet from moving (i.e. rolling on the surface as the microfluidic device or chip moves around or even turns over) during manipulation. Of course, these movements are considered without violent shaking. A drop that is found to be stable when a particular engagement procedure is used may be found to be unstable when another procedure (e.g., a more vigorous procedure) is used.

“Controlled engagement” means proper alignment and flatness of the path or port. Refers to the engagement of two devices, allowing both drop-to-drop connections without resulting in loss of drop stability. For example, if the device is held firmly in place or meets before engaging the drop on the opposite device, drop stability will be compromised.

General description of the invention

In one embodiment, the present invention relates to a microfluidic device, e.g., in vivo, connected (preferably removably) to a perfusion manifold assembly such that fluid enters a port of the microfluidic device from a fluid reservoir at a controllable flow rate, optionally without tubing. [00112] [0012] [0011] [0011] [0012] [0012] [0012] [0012] Consideration is given to a perfusion manifold assembly that allows perfusion of an organ microfluidic device on a chip containing cells that mimic cells in the organ or that mimic at least one function of the organ. In one embodiment (as shown in Figures 1A, 1B and 1C), the perfusion manifold assembly (10) comprises i) a cover or shroud (11) configured to act as a top of one or more fluid reservoirs, ii) one a fluid reservoir (12) above, iii) a capping layer (13) beneath said fluid reservoir(s), iv) a fluid backplane (14) comprising a resistor beneath and in fluid communication with said fluid reservoir(s), and v) a protruding member or skirt (15) for engaging a microfluidic device (16) or chip, preferably positioned on a carrier (17), said chip having at least one microchannel (1) and In fluid communication with more than 2 ports. The assembly may be used with or without a lid or cover. Other embodiments (discussed below) lack skirts or protruding members. In one embodiment, the carrier 17 includes a tab or other gripping platform 18, a retaining mechanism such as a clip 19, and a visualization cutout 20 for imaging the chip. The cutout 20 allows installation of the carrier (e.g., a carrier engaged or disengaged with a perfusion manifold assembly or “pod”) onto a microscope or other inspection device, eliminating the need to remove the chip from the carrier. You can observe the chip without it. In one embodiment, the fluid resistor includes a series of switchbacks or tortuous fluid channels. Figure 32 shows an improved schematic diagram of one embodiment of a backplane, showing fluid resistor channels 32a and chip engagement reliefs 32c or ports. As described in more detail in U.S. Provisional Application Serial Nos. 62/024,361 and 62/127,438, which became PCT/US2015/040026, and are incorporated herein by reference, particularly the discussion of resistors, resistor design, and pressure. Various fluid resistor designs are considered. In one embodiment, the perfusion manifold assembly is made of plastic and is disposable, i.e. docked into and perfused with a microfluidic device and then disposed of. Although the present invention contemplates a “disposable” embodiment, the members are (alternatively) reusable (eg, for cost considerations).

In one embodiment, the microfluidic device (e.g., chip) 16 may be first installed in the carrier 17 (e.g., chip carrier) prior to engaging the perfusion manifold assembly 10, or It can be directly engaged with. In either case, the (optional) removable connection of the microfluidic device to the manifold must either a) prevent air from entering the microchannel, or b) provide a way for undesirable air to be removed or vented from the system. . In fact, air removal may be necessary in some embodiments both during chip attachment and during microfluidic device use.

In one embodiment to prevent air from entering the microchannels, the microfluidic device is detachably connected using a “drop-to-drop” “chip-to-cartridge” connection. In this embodiment, the injection port of the microfluidic device has a droplet 22 protruding therefrom (FIG. 15A), and the surface of the perfusion manifold assembly or “cartridge” 10 for engaging the device has a corresponding droplet ( 23). After the two come together (Figure 15b), the droplets fuse, allowing fluid communication without the introduction of air into the channel. In one embodiment, the chip carrier is designed so as not to interfere with the “drop-to-drop” connection. For example, the carrier surrounds the sides of the microfluidic device in one embodiment, but does not surround its bonding surface 21. Figure 15A shows the perfusion manifold 10 without a skirt, and it should be noted that the microfluidic device or chip engages underneath (rather than on the side) of the perfusion manifold.

It is not intended that the present invention be limited to only one way to detachably connect a microfluidic device. In one embodiment, the microfluidic device, such as an organ microfluidic device on a chip that mimics one or more functions of cells in an organ in the body or that includes cells that mimic at least one function of an organ, has a droplet (22) protruding upward. The assembly is preferably approached from the side (Figure 16a) or from below (Figure 16b), while the corresponding drop 23 on the assembly (or other type of fluid source) protrudes downward. The microfluidic device (or device carrier) may include a portion 24 configured to engage a side track 25 or other guiding mechanism. In another embodiment, a microfluidic device, such as an organ-on-a-chip microfluidic device that mimics cells in an organ in the body or that includes cells that mimic at least one function of an organ, allows the droplet to project downward and onto the assembly from above. While approaching, the corresponding droplet on the assembly protrudes upward. In yet another embodiment, a microfluidic device, such as an organ microfluidic device on a chip comprising cells that mimic cells in an organ in the body or mimic at least one function of an organ, approaches the assembly from the side and includes a hinge, It is positioned by a pivot (Figure 16d, see arrow) at the socket, or other pivot point 26. In yet another embodiment, the microfluidic device engages in the manner of an audio cassette or CD such that the droplets project upwards, while the corresponding droplets on the assembly project downwards, where lateral and upward movements are combined ( Figures 16b-16c).

In one embodiment, the microfluidic device 16 is detachably connected to the perfusion manifold assembly 10 by a clipping mechanism that temporarily “locks” the microfluidic device, such as an organ on a chip, in place (FIG. 4A, 4b, 4c and 4d). In one embodiment, clipping or “snap fitting” involves a protrusion on the carrier 19 that acts as a retention mechanism when positioning the microfluidic device 16. In one embodiment, the clipping mechanism is similar to the interlocking plastic design of a LEGO™ chip and includes a straight-down clip, a friction fit, a radial-compression fit, or a combination thereof. However, in another embodiment, the microfluidic device, or more preferably the carrier 17 containing the microfluidic device 16, is provided with guide rails, side slots, inner or outer tracks 25, or (e.g. The clipping mechanism is triggered only after engaging the perfusion manifold assembly (or cartridge) on another mechanism that provides a stable glide path for the device as it is delivered into position (either by machine or by hand). Guide rails, side slots, inner or outer tracks (25) or other mechanisms may, but need not, be strictly linear and may be located on skirts (15) or protruding members attached to the main body of the flow manifold assembly (10). You can. In one embodiment, the starting portion of the guide rail 25 (or side slot, internal or external track or other mechanism) is linear, followed by an angled slide 27 which provides a larger opening for easier initial positioning. or an essentially linear portion (28). In one embodiment, the distal portion 29 (close to the corresponding port of the assembly) of the otherwise linear (or essentially linear) guide rail 25 (or side slot, internal track or other mechanism) is angled upward. (or curved) (Figure 16b), a combination of linear movements (e.g., initial) and upward movements are used to achieve the connection.

In some embodiments, it is important that the droplets remain in place in their corresponding fluid ports despite any period of movement or flipped orientation of their substrates. It is also desirable for the droplets to maintain their size so that, for example, the drop-to-drop process is constant regardless of the speed of the engagement process. Accordingly, the present invention contemplates designs and methods that provide stable drops. Stable dripping is considered for aqueous as well as non-aqueous liquids. Although the present inventors focus on embodiments of the invention without loss of generality to aqueous drops, those familiar in the art will be able to adapt the above embodiments, especially the use of hydrophilic and hydrophobic regions or materials based on the wetting properties of the liquid. Must be able to. In some embodiments, a drop may be confined within a first region of the substrate by surrounding the first region with a second region, where the second region is hydrophobic (or more generally has a hydrophobic property that resists wetting by liquid of the droplet). tend to). The second region includes selection of one or more hydrophobic materials (e.g. PTFE, FEP, certain grades of nylon, etc.), surface treatment (e.g. plasma treatment, chemical treatment, ink treatment), gasket (e.g. The substrate may be rendered hydrophobic by the use of (e.g., films, o-rings, adhesive gaskets), masking during processing of at least one other area of the substrate, or a combination thereof. In some embodiments, the drop can be confined within a first region of the substrate by surrounding the first region with geometric features. In some embodiments, the geometric features may be ridges or depressions. Without being bound by theory, these features restrict the droplet using their edges to interact with the surface layer of the droplet (and correspondingly the surface tension of the droplet), for example by "pinning" the surface of the droplet. It can work to do so. In some embodiments, the droplet may be limited to cover a first area of the substrate by adapting the first area to be hydrophobic (or more generally prone to wetting by the liquid of the droplet). The first region includes selection of one or more hydrophilic materials (e.g., PMMA, PLA), surface treatment (e.g., plasma treatment, chemical treatment, ink treatment), gasket (e.g., film, o -rings, adhesive gaskets), masking during processing at least one of the other areas of the substrate, or a combination thereof.

In one embodiment, the bonding surface 21 (or at least a portion thereof adjacent to the port opening) of the microfluidic device is hydrophobic or rendered hydrophobic (or protected with a mask during plasma treatment to prevent it from becoming hydrophilic). In one embodiment, the mating surface of the perfusion manifold assembly or cartridge (or at least a portion thereof adjacent the port opening) is hydrophobic or rendered hydrophobic (or protected with a mask to prevent it from becoming hydrophilic during plasma processing). In one embodiment, both the bonding surface of the microfluidic device (or at least a portion thereof adjacent to the port opening) and the bonding surface of the perfusion manifold (or at least a portion thereof adjacent to the port opening) are hydrophobic or rendered hydrophobic (or treated with plasma). (Prevent it from becoming hydrophilic by protecting it with a mask).

An advantage of the carrier is that it does not need to contact the surface of the microfluidic device during the detachable connection with the perfusion manifold assembly. The carrier may have a plate, platform, handle, or other mechanism for gripping the carrier 18 without contacting the mating surface 21 of the microfluidic device 16. Retention mechanism 19 may include a protrusion, hook, latch or lip that engages with one or more portions of the perfusion manifold assembly and, more preferably, with a skirt of the perfusion manifold assembly to provide a “snap fit.”

In other embodiments (FIGS. 27, 28, and 29), one or more gaskets can be used to evacuate air (e.g., any air introduced due to the removable connection of the microfluidic device and perfusion manifold assembly). . In one embodiment, the bubbles can be trapped (thereby limiting their impact), and in an alternative embodiment, the bubbles can be vented. One method involves using hydrophobic exhaust material (molded or sheeted). For example, the hydrophobic exhaust material may include PTFE, PVDF, hydrophobic grades of nylon, or combinations thereof. In some embodiments, venting can be achieved by using materials that exhibit high gas permeability (e.g., PDMS). In other embodiments, venting is achieved by using porous materials, such as sintered materials, porous membranes (e.g., track-etched membranes, fiber-based membranes), open-cell foams, or combinations thereof. It can be. In a preferred approach, air is released from the vented (or being vented) gasket. In some embodiments, the perfusion manifold assembly or microfluidic device includes an exhaust port adapted to provide a passageway for unwanted gases to escape.

Once the microfluidic device (or “chip”) is docked to the perfusion manifold assembly, the assembly-chip combination is placed in an incubator 31 (typically set at a temperature above room temperature, e.g., 37° C.), or more preferably Can be installed in a culture module 30 capable of supporting a plurality of assembly-chip combinations, the culture module being configured to fit on an incubator shelf (see Figure 5). This makes it possible to easily handle multiple (e.g., 5, 10, 20, 30, 40, 50 or more) microfluidic devices at a time. For example, if a culture module contains 9 assembly-chip combinations and the incubator is sized for 6 to 9 culture modules, 54 to 81 “organs on a chip” can be handled in a single incubator. (Figures 5 and 8). In another example, if a culture module includes 12 assembly-chip combinations and the incubator is sized for 4 to 6 culture modules, then 48 to 72 “organs on a chip” can be handled in a single incubator. there is. The perfusion manifold can be easily removed and inserted into the culture module without destroying the fluidic connection to the chip. In one embodiment, the culture module may be maintained at a temperature above room temperature, such as 37° C., even when not placed in an incubator.

In one embodiment (FIG. 6), the culture module 30 provides movement of various members together with a removable tray 32, pressure surface 33, and pressure controller 34 for positioning the assembly-chip combination. Includes optional user interface 46 for control. In one embodiment, tray 32 can slide. In one embodiment, the tray is placed on the culture module and the tray is moved up through the tray mechanism 35 to engage the pressure surface 33 of the culture module, i.e. the cover or cover of the perfusion manifold assembly 10. (11) engages the pressure surface of the culture module (30). Multiple perfusion assemblies 10 can be attached to the pressure controller by a single operation by the tray mechanism. In another embodiment, the tray is positioned on the culture module and the pressure surface of the culture module 30 is moved down to engage the tray 32, i.e., the cover or shroud 11 of the perfusion manifold assembly 10. do. In either case, in one embodiment ( FIGS. 2A and 2B ), the cover or lid comprises a port, such as a through-hole port (36), that is engaged by a corresponding pressure point on the pressure surface (33) of the culture module. These ports (36), when engaged, transmit the applied pressure inwardly through the cover and through the gasket (37) and apply pressure to the fluid in reservoir (12) of the flow manifold assembly (10). Therefore, in this embodiment, pressure is applied to the reservoir(s) through the lid 11 and the lid seal. For example, when 1 kPa is applied, this nominal pressure produces a flow rate of approximately 30-40 uL/hr in one embodiment. Alternatively, these ports 36 move inwardly on the cover to contact the gasket when engaged (i.e., the ports essentially act like plungers).

8A is a schematic diagram of another embodiment of a culture module 30, showing trays (or racks) 32 and sub-trays (or nests) for transporting and inserting perfusion disposables 10 into the culture module. , the culture module has two openings 48, 49 in the housing for receiving trays, and a user interface 46 for controlling the process of perfusion disposable engagement and pressure application. A typical incubator (not shown) can support no more than six modules (30). Figure 8B is a schematic diagram of the same embodiment as Figure 8A, but with two trays (or racks) 32 housing a pressure module 30 with a user interface 46 (e.g., LCD screen) for process control. It is inserted into the two openings (48, 49) of (53).

9A shows the pressure manifold (50) in the open position and the tray or rack (32), sub-tray or nest (47), and irrigation disposables (10) positioned below the pressure manifold (50). This is a schematic diagram of the interior (i.e., the housing has been removed) of one embodiment of a module, which is not engaged (with sufficient clearance to remove them), and the actuating assembly 51 includes a pneumatic cylinder 52 thereon.

9B shows the pressure manifold 50 in a closed position and the tray or rack 32, sub-tray or nest 47, and irrigation disposables 10 positioned below and engaged with the pressure manifold 50. is a schematic diagram of the interior (i.e., with the housing removed) of one embodiment of a module, where the actuating assembly 51 includes a pneumatic cylinder 52 thereon. A pressure manifold (50) engages all perfusion disposables (10) simultaneously while medium perfusion is desired or required. Independent control of flow rates in the upper and lower channels of chip 16 can be achieved. Pressure manifold 50 can be disconnected (without complex fluid separation) to remove tray 32 or nest 47 for imaging or other tasks as needed. In one embodiment, pressure manifold 50 can be separated from multiple flow manifold assemblies simultaneously. In one embodiment, the irrigation disposables (10) may not be rigidly secured to the nest (47), but rather they may be positioned proximate to the pressure manifold (50). In a preferred embodiment, an integrated alignment feature in the pressure manifold (50) provides a guide for each irrigation disposable (10).

In one embodiment, the cover or lid is made of polycarbonate. In one embodiment, each through-hole port is associated with a filter 38 (e.g., a 0.2um filter). In one embodiment, the strainer is aligned with holes 39 in a gasket located underneath the cover.

A culture module comprising a pressure manifold is contemplated, allowing perfusion and optionally mechanical actuation of one or more microfluidic devices, such as an organ microfluidic device on a chip containing cells that mimic the function of at least one organ in the body. do. 10A is a schematic diagram of one embodiment of a pressure manifold 50, showing a PD engagement surface 54 with several PD engagement positions (in this case six engagement positions). 10B is a close-up of the engagement surface 54 of the pressure manifold 50, highlighting the spring shuttle 55, valve seal 56, and alignment feature 57 (which allows the PD to align with the manifold). Show the part that has been done. The spring shuttle is an optional means that allows the pressure manifold to sense the presence of a PD at a particular PD engagement location. In certain embodiments, the presence of a PD depresses a spring shuttle, which opens one or more valves disposed within the pressure manifold, allowing application of pressure or fluid flow to the PD. Additionally, in the absence of PD, the shuttle is not depressed and the valve remains closed; This is intended to prevent pressure or fluid leaks. The valve seal shown is adapted to form a pressure and/or fluid seal to the corresponding features in the PD and to the pressure shroud, if present. 10C shows the PD engagement surface 54 along an enlarged portion highlighting the shroud compressor 58, valve seal 56, and alignment feature 57 (allowing the PD to align with the manifold). A schematic diagram of another embodiment of a pressure manifold 50. The shroud compressor may apply a force on the pressure shroud to assist in establishing pressure and/or maintaining a fluid seal between the pressure shroud and the reservoir. In one embodiment, the shroud compressor comprises a spring, an elastomeric material, a pneumatic actuator, or a combination thereof, selected and sized to apply a force commensurate with the force required to maintain the pressure and/or fluid seal. You can have 10D is a side schematic diagram of one embodiment of a pressure manifold 50, showing the PD engagement surface 54. FIG. 10E is a schematic diagram of one embodiment of a pressure manifold 50, showing the opposing surface 67. 10F shows one spring carrier (70) and spring (71) (among many) removed from the manifold body, one seal (72) (among many), shuttle (73) and valve body. One embodiment of a pressure manifold (50) showing the PD engagement surface (54) with the PD guide (68) and lower backer plate (69) removed, highlighted by showing (74) removed from the manifold body. This is a schematic diagram. The external spring 75 adapted to press the pressure manifold against the irrigation disposable is also highlighted by showing it removed. 10G is a schematic diagram of one embodiment of a pressure manifold 50, showing the opposing surface 67 (not the PD engagement surface) with the upper backer plate 76 and capping strip 77 removed. A trans-manifold channel 78 is shown adapted to derive and optionally distribute pressure and/or fluid from one or more pressure ports. Additionally, one screw 79 (out of several) and one gas port 80 (out of five, including both gas and vacuum ports) are shown by showing them removed from the manifold body 50. 10H is a schematic diagram of one embodiment of a pressure manifold 50, showing a top view of the manifold via channel 78 and one of several ports 81. The via channels can be created using a number of methods known in the art, such as forming, machining, ablating, lamination, 3D printing, photolithography, and combinations thereof.

11A shows one embodiment of a valve 59 (Schrader valve) in a pressure manifold 50, showing the silicone membrane 60, shuttle 61, air inlet 62 and cover plate 63. This is a schematic diagram. In this embodiment, a spring shuttle is integrated into the valve and adapted to actuate the valve by pressing the poppet of the Schrader valve. FIG. 11B is a side view photograph of one embodiment of a valve for a pressure manifold, showing the valve seat 64 and membrane 60 acting as a valve seal, and FIG. 11C is a top view photograph thereof. 11D is a schematic interior side view of one embodiment of a pressure manifold 50, showing a plurality of valves 59, poppets 65, valve seals 66, and PD covers 11 in the manifold body. am. In operation (to engage the PD), the valve seal 66 is deflected by the displacement of the poppet 65.

12A shows one embodiment of a connection scheme comprising a tubing connection manifold 82 allowing four culture modules 30 (three shown) to be connected inside a single incubator 31 using one or more hub modules. Schematic diagram of the embodiment (the two circles provide an enlarged view of the first end 83 and the second end 84 of the connection). FIG. 12B is a photograph of the gas hub and vacuum hub (collectively 85) along tubing 86 for the connection shown in FIG. 12A. Although this connection method is arbitrary, it provides a convenient way to use multiple culture modules in a single incubator.

DETAILED DESCRIPTION OF THE INVENTION

A. Pressure cover

The present invention in one embodiment contemplates a “perfusion manifold assembly” or “perfusion disposable” that facilitates culturing of organs on a chip within a culture device. Although the present invention contemplates a “disposable” embodiment, the members may (alternatively) be reused (e.g., for cost considerations).

In one embodiment, these perfusion disposables (PDs) include one or more inlet and one or more outlet reservoirs, as well as the necessary elements for pumping. In particular, in embodiments of the invention the perfusion disposables include one or more resistors used for pressure-driven pumping (see Figure 32A). In pressure-actuated embodiments, the equipment creates or controls fluid flow by applying pneumatic pressure (whether positive or negative) to one or more reservoirs. One advantage of this approach is that the pressure-actuated design avoids liquid contact with the equipment, which provides benefits in terms of sterility and ease of use (e.g., avoiding gas bubbles in liquid lines). In some embodiments, the equipment applies pressure directly to one or more reservoirs (without a cover). A sufficient pressure seal can be achieved by one or more gaskets integrated on the perfusion disposables and/or equipment (e.g., as part of a pressure manifold). However, when the perfusion disposables are external to the equipment, it is desirable for the reservoir to be protected from contamination, for example from environmental particles or airborne microorganisms. Accordingly, in the same embodiment, a user may provide a cover that can be used to cover the reservoir when outside the equipment and/or a substrate that transmits pressure but blocks contaminants (e.g., a suitable material disposed over an opening in the reservoir). It may be desirable to use a PD embodiment comprising a filter). However, these solutions typically suffer from drawbacks. In particular, if the user is expected to install the cover, it is required that the user maintain the cover while the perfusion disposable is engaged with the equipment, and ideally install the cover as soon as the PD is disconnected from the equipment; in most situations, , these actions have a negative impact on the user experience. Additionally, strainers placed over the openings of the reservoir typically block access to the reservoir by pipettes and other typical laboratory tools, negatively limiting their ease of use.

According to aspects of the invention, the inventors have discovered that a microfluidic device or a device adapted to receive a microfluidic device (e.g., a perfusion disposable) can be placed on the device even while engaged with the device. A “cover” is disclosed, wherein the pressure cover is adapted to allow pressure communication between the equipment and the device. The present invention, in some embodiments, the pressure cover is a removable cover adapted to be placed on one or more reservoirs of a microfluidic device or a device adapted to receive a microfluidic device (e.g., a perfusion disposable), and Provided that the cover includes at least one equipment-facing port and at least one reservoir-facing port, wherein the pressure cover is adapted to transfer pressure between at least a portion of the equipment-facing port and at least a portion of the reservoir-facing port. do. In some embodiments, the pressure shroud includes at least one “through hole” port, an opening connecting first and second surfaces of the shroud, the opening on the first surface being adapted to form an equipment-facing port; The opening on the second surface is adapted to form a reservoir-facing port. In some embodiments, the through-hole port is circular, rectangular, triangular, polygonal, straight, curved, oval, and/or curved. However, in some embodiments, the lid includes a channel connecting at least one equipment-facing port and at least one reservoir-facing port, where the ports cannot be placed directly opposite each other. This embodiment may be useful, for example, when there is a need to adapt between equipment access locations and reservoir locations, for example when the same equipment is desired to support the operation of multiple types of perfusion disposables.

In some embodiments, the pressure shroud is adapted to form a pressure seal between the pressure shroud and at least one reservoir. In some embodiments, the pressure cover engages at least one reservoir forming a cover-to-reservoir pressure seal. In some embodiments, a pressure shroud is adapted to form a pressure seal between the pressure shroud and at least one piece of equipment. In some embodiments, the pressure cover engages at least one device forming a cover-to-equipment pressure seal. Any lid-to-reservoir seal and lid-to-equipment seal may utilize any sealing method known in the art, such as face seal, radial seal, tapered seal, friction bond, or combinations thereof. Can be selected from the group. Any of the above seals may utilize one or more gaskets, o-rings, elastic materials, flexible materials, adhesives, sealants, grease, or combinations thereof. The present invention is not intended to be limited to designs with complete pressure sealing, as complete pressure sealing may not be necessary. Rather, a certain amount of gas leakage can be tolerated because the equipment can actively adjust pressure to compensate for the leak. By relaxing the requirement to obtain a complete seal on one or both sides, the design can be simplified and costs reduced.

In some embodiments, the pressure shroud includes a load concentrator. For example, in some embodiments, the pressure shroud includes a ridge surrounding at least one equipment-facing port. In some embodiments, the pressure shroud includes a ridge surrounding at least one reservoir-facing port. It is known in the art that such load concentrators can act to improve pressure seals by improving reliability or reducing the forces required, and designs known in the art include, for example, rectangular, semicircular, triangular, trapezoidal. and polygonal ridges. Therefore, the lid-to-equipment pressure seal can be improved by using a load concentrator surrounding the equipment-facing port, and the lid-to-reservoir pressure seal can be improved by using a load concentrator surrounding the reservoir-facing port. there is.

In some embodiments, the pressure cover includes a strainer. For example, the pressure cover can include membrane filters, sintered filters, fiber-based filters, and/or track-etched filters. In some embodiments, the filter is disposed within or adjacent to one of the through-hole ports and/or openings thereof. In some embodiments, the strainer is disposed within or adjacent to one of the channels included in the lid and/or openings in the channels.

In some embodiments, the filter is selected to improve the sterility of the reservoir and/or block particles, contaminants, or microorganisms. In some embodiments, the filter is characterized by an effective pore size of 0.4 um or less, 0.2 um to 2 um, 1 um to 10 um, 5 um to 20 um, 10 um to 50 um. It is known in the art that filters featuring an effective pore size of 0.4 um or less are desirable for maintaining sterility. However, filters such as Porex 4901 with a 25um effective pore size were found to be effective in maintaining sterility.

In some embodiments, the pressure cover includes one or more gaskets. In some embodiments, one or more gaskets are adapted to allow or improve pressure sealing (which may nevertheless not be a complete seal). In some embodiments, at least one gasket is disposed on the reservoir-contacting surface of the lid. In some embodiments, at least one gasket is disposed on the equipment-contacting surface of the cover. In some embodiments, the gasket is adapted to allow or improve pressure sealing with multiple reservoirs. In some embodiments, gaskets are adapted to allow or improve pressure sealing at multiple equipment-facing ports. In some embodiments, the one or more gaskets include elastomers, flexible materials, o-rings, and/or combinations thereof. In some embodiments, one or more gaskets are formed by extrusion, casting, injection molding (e.g., reaction-injection molding), die cutting, and/or combinations thereof. In some embodiments, the at least one gasket can be glued (e.g., using an adhesive tape), clamped, screwed down, bonded, heat staked, welded (e.g., ultrasonically, by a laser), fused (e.g. (e.g., solvent-assisted bonding) and/or combinations thereof.

For example, one of the inventive embodiments of the cover includes ports 5 that allow pneumatic (e.g. vacuum) control of the (arbitrary) chips connected to communicate through the cover (Figure 2a -see 2e). It is not intended that the cover be limited to communication only by pneumatic pressure, but it is contemplated that the cover may additionally communicate with fluid or electrical connections.

In one embodiment, the cover can include a sensor. For example, the cover may include a pressure sensor, for example to measure the pressure occurring on one or more reservoirs. Additionally, the cover may include liquid-level sensing to determine the amount of liquid present in the reservoir or whether a particular filling (or depletion) threshold has been passed. This can be done in a variety of ways. In one embodiment, a detection liquid that optically utilizes a difference in refractive index is contemplated. In this embodiment, the air-filled compartments and channels disperse the light, while the liquid or fluid-filled channels focus the light. More specifically, the refractive index of liquid is between 1.3 and 1.5, whereas the refractive index of air is only 1.0. In one embodiment, each optical detector consists of a pair of IR emitters (SEP8736, 880 nm, Honeywell) and a phototransistor (SDP8436, 880 nm, Honeywell). In this embodiment, IR is selected over visible light because it is less sensitive to interfering light.

The ability to easily remove fluid from various reservoirs (e.g., take samples, replenish media, add test agents, etc.) is a desirable feature. A particularly desirable feature is that it allows the use of standard laboratory pipettes and syringes for this task. However, such fluidic access (especially using pipettes) requires that the accessed reservoir be open to the environment. This is undesirable, especially when chips or disposables are transported or used outside of the equipment, as the openings may provide a means for contamination of the reservoir. A typical solution to this problem is to include covers that can be applied to one or more bins when they are not being accessed. However, simply including a cover can complicate the use of the technology because the user typically must not only actively install and remove the cover, but also maintain the cover in a sterile manner near the equipment.

One solution is to include means to automatically remove and/or install the cover as part of the system (whether integrated into the culture equipment or in a separate module). For example, the system may include a mechanical actuator that can engage a cover installed on a deployed irrigation disposable and remove it prior to engagement with the pressure system. This mechanical actuator allows reinstallation of the cover upon removal of the irrigation disposable. In an alternative embodiment, the system comprises means for applying a cover to a perfusion disposable product before or upon removal of the cover, for example, a cover originating from a magazine of stored covers.

A disadvantage of systems with means to automatically remove and/or install covers (discussed in the previous paragraph) is that they require one or more mechanical actuators which can make the task difficult in practice. Additional challenges include: the design of the reservoir and especially its openings, which require liquid access (manual sampling or replenishment using pipettes), pressure-actuated systems (ensuring a good pressure seal to the equipment, for example) and manufacturing. It aims to satisfy the needs for (e.g. injection molding of reservoirs). In practice, these requirements may conflict with each other. For example, manual access may require a wide reservoir opening, but in contrast it may be desirable to have a narrower pressure connection to reduce forces on the equipment.

A better solution disclosed herein is to include a “pressure cover” (see FIGS. 2A, 2B, 2C and 2D). This pressure cover can be installed on the reservoir to reduce the possibility of contamination and is designed to remain virtually in place while the perfusion disposable is engaged with the equipment. To ensure that it remains substantially in place while engaging the equipment, the cover preferably includes a) one or more features designed to interface with the equipment (e.g., to receive positive or negative pressure), and b) (e.g., one or more features designed to interface with one or more reservoirs (to create a pressure seal or minimize gas leakage so that pressure is applied to the reservoir), and c) at least a portion of feature (a) and feature (b) ) includes means for pressure communicating with at least a portion of the The pressure cover or portion thereof may be transparent or translucent. This may make it possible to view the liquid level in the reservoir, for example. The pressure cover may include markings indicating the nature or name of each reservoir.

In one embodiment of the pressure cover, the opening in the pressure cover (e.g., at the top thereof) may be smaller than the reservoir to reduce the surface area open to contaminants and/or reduce the area subject to pressure sealing. there is. In another embodiment, the lid may include a strainer or multiple strainers 38 to prevent solids and particles from entering (see Figure 2A). For example, the lid may include a 0.2um or 0.4um filter, which is known to reduce the entry of bacteria and other contaminants. Several materials and technologies are available for such filters. Since the filters need to transmit only pressure and not liquid, for example track-etched filters (e.g. PTFE, polycarbonate, PET), paper filters, porous and expanded materials (e.g. cellulose and derivatives, polypropylene, etc.), sintered materials (e.g., Porex filters) can be used.

In one embodiment, the cover may include means to allow gas flow but little liquid flow. Examples include hydrophobic porous membranes or filters, gas permeable membranes or filters, etc. This approach can also help reduce the likelihood of leaks.

In one embodiment, the cover can include a deformable portion that can be deformed to transmit pressure. For example, this may be an elastic or plastic membrane that leads to a reservoir as positive pressure is applied. Similarly, the cover may include a plunger used to transfer pressure from the equipment to one or more reservoirs. Because the membrane or plunger may apply a reverse force, care must be taken to ensure that the desired pressure is applied to the interior of the reservoir. This, for example, a) ensures that the reverse force is small or understood through the design of the membrane, plunger or operating pressure range, b) measures the pressure inside the reservoir and uses this to control the applied pressure, and c) ensures that the applied pressure is controlled. This can be done by monitoring the generated flow to control the pressure. The deformable part provides a way to communicate pressure.

One side of the pressure shroud (equipment-facing or perfusion disposable-facing) as well as each opposing surface (equipment and perfusion-disposable features that interact with the pressure shroud) may be designed to enable pressure sealing in a number of different ways. You can. In one embodiment, the present invention contemplates one or more regions comprising one or more elastic or flexible materials. In one embodiment, it is attached to one or more gaskets (see Figure 2A), which may be made of, for example, elastomeric or flexible materials (e.g., silicone, SEBS, polypropylene, Viton, rubber, etc.). is carried out by Gaskets can be formed in a variety of ways, such as cut into flat sheets, o-rings (not necessarily round or cross-sectioned), etc. In one embodiment, this is accomplished by one or more ridges acting as load concentrators (see Figure 2C). Without wishing to be bound by theory, they act to localize the sealing force and create an elevated localized sealing pressure. These ridges potentially engage a gasket or flexible material on the opposing surface. Care must be taken to design the shape of the ridges (particularly those that are shaped to engage the opposing surfaces), as this may have a substantial effect on the sealing pressure required. Various shapes (e.g., rectangular, triangular, trapezoidal, semicircular or circular sections, etc.) are contemplated. In one embodiment, the sealing tooth has a trapezoidal shape for improved sealing (see Figure 2C). Alternatively, the gasket may be incorporated into the reservoir or lid in the form of an overmolded elastomer (e.g., silicone, SEBS, etc.). The overmolded elastomer itself then has a suitable shape (eg, a tooth or o-ring semicircular section) to act as a seal.

The approach need not be limited to a single design. In one embodiment, the present invention contemplates a combination of one or more regions comprising one or more elastic or flexible materials. Moreover, the gasket or ridge can be carried along the reservoir, isolated from each other in terms of applied pressure, or can comprise two or more reservoirs, which can reduce complexity. In one embodiment (see Figure 2D), a passageway surrounds all chambers of the reservoir chamber-cover assembly seal, isolating each chamber from the other. In one embodiment (see Figure 2D), there are two reservoirs, each of which is an injection chamber (6a, 6b) and an evacuation chamber (7a, 7b), and a separate reservoir capable of delivering vacuum to the chip or other microfluidic device. It has an (optional) vacuum chamber (8). In one embodiment (FIG. 2E), the reservoir chamber-cover assembly seal includes sealing teeth (9).

The present invention is not intended to be limited to designs with complete pressure sealing, as complete pressure sealing may not be necessary. Rather, a certain amount of gas leakage can be tolerated because the equipment can actively adjust pressure to compensate for the leak. By relaxing the requirement to obtain a complete seal on one or both sides, the design can be simplified and costs reduced.

The pressure cover can be secured or placed on the reservoir (whether on a perfusion disposable or directly on the chip) in a variety of different ways. Embodiments are where the liquid or gas seal between the lid and the reservoir(s) is even external to the equipment (e.g., the lid may be held securely in place by something other than the equipment), and the seal is of the equipment. These may include those produced by action (e.g., the device presses the lid against the reservoir during perfusion). In another embodiment, the invention contemplates a combined approach, for example, as in the first option above, the lid is designed to create at least a partial seal, but as in the second option above, the seal is formed by the action of the equipment. approved or guaranteed. An advantage of approaches that provide at least some degree of sealing of covers even for reservoirs external to the equipment is that they can reduce the risk of spills and contamination (e.g., due to handling or transport).

Examples of approaches for securing or placing a pressure sheath (regardless of which of the three approaches above) include: a) the sheath can be simply placed over the reservoir or irrigation disposable (this can be done by simply placing the overhanding portion of the sheath); Because it can be assisted by , the cover cannot simply be slid off); b) Covers can be screwed, glued or peened in place; and c) the cover can be clipped in place. In an alternative embodiment, it may also be held down by a spring, for example a hinged lid having a spring that applies a force to close the lid.

Clip features may be present on a sheath, irrigation disposable, chip, or combinations thereof. Additionally, some embodiments use a separate substrate that provides a clipping member (i.e., a separate piece to clip the lid in place). The advantage of the clipping approach is that it can facilitate easy application and removal of the cover while still holding the cover in place. Clipping may be arbitrary, for example it may be applied when transporting or transporting the device, or it may be ignored during regular use.

In some embodiments, the cover is asymmetrical or includes a lock-and-key feature to ensure that the cover is correctly oriented relative to the perfusion disposable product and/or equipment.

Several features of a perfusion disposable product (PD) could potentially be included in the “chip” itself or in a different device coupled to the chip. If the reservoir is contained within a chip, for example, a pressure shroud directly on top of the chip may be used.

Although the pressure shroud is discussed above in relation to pressurizing one or more reservoirs within a perfusion disposable or perfusion manifold assembly, the pressure shroud is not intended to be limited to use only in these embodiments. In fact, it is contemplated that the pressure sheath may be used with other microfluidic devices. The pressure cover may be movable or removably attached to another microfluidic device to allow improved access to the member (e.g., reservoir) from within. The pressure cover can be removed from these other devices and the other devices can be used without the cover. In one embodiment, the other microfluidic device comprises cells on a membrane and/or in or on one or more microchannels.

B. Tray system

It is desirable to be able to remove the chip and/or perfusion disposables from the equipment without having to remove the equipment itself, for example from the incubation area. It is also desirable to be able to remove groups of chips and/or irrigation disposables together. This is because the operations performed on the chip/disposable (e.g., media replenishment, agent administration, sample collection) often have to be performed in batches, regardless of whether they are performed automatically or manually. . For example, it is convenient to remove a group of chips/disposables at once only if this will facilitate transport to a biosafety cabinet or culture hood.

To address this need, in one embodiment, the present invention contemplates a system in which perfusion disposables can be inserted into or removed from equipment (or modules) in groups by a tray system (see Figure 6). For example, this embodiment allows each equipment to accommodate two trays (or racks) with six perfusion-disposables each (8a and 8b).

In one embodiment, the tray (or rack) 32 provides alignment of the perfusion disposables 10 and the equipment 30 (e.g., alignment of reservoir or port locations with corresponding pressure or fluid connections included in the equipment). It can be done easily. This can be done in a number of ways, such as by providing positioning features for the perfusion disposables (or any additional members holding them) within the tray and positioning features for the trays and alignment features for the perfusion disposables within the equipment (57). This can be performed by providing (see Figure 10b). Features that can be used to support this alignment include reference surfaces to facilitate registration, pins, guides, shaped surfaces (e.g., fillets and/or chamfers), springs or elastic members, etc. This is included. These may be included in trays, equipment, perfusion disposables, or combinations thereof.

The tray may optionally be designed to capture leaks originating from perfusion disposables or equipment connections. The tray may optionally include one or more optical windows that may facilitate microscopic observation or inspection. This allows the tray to be mounted on a microscope or other inspection device, allowing the chips to be observed without the need to remove each disposable item from the tray. Correspondingly, the tray can optionally be designed to minimize the imaging distance, for example by lying flat on or fitting into the microscope stage. The system may optionally include means for maintaining one or more irrigation disposables within the tray. For example, a perfusion disposable product can be clipped to a tray, and the clip features can be present on the perfusion disposable product, the tray, an additional substrate, or a combination thereof.

In some embodiments, the tray system includes one or more sub-trays (or nests) 47 that fit into a carrier tray 32 (see Figure 8A). The sub-tray allows a subset (e.g., three) of perfusion disposables to be removed from the tray simultaneously. This may be useful, for example, if one or more operations performed on the chips/disposables would benefit from a small amount of chips present in the carrier tray. For example, in some instances, we prefer to install no more than three disposables at a time on the microscope stage, minimizing the time the chips/disposables spend outside their preferred incubation and perfusion environment. As a result, this embodiment includes a carrier tray 32 supporting two sub-trays 47, each sub-tray supporting three perfusion disposables 10 (see Figure 8A).

The sub-tray can facilitate aligning perfusion disposables with respect to the equipment. This can be accomplished in a number of ways, such as by providing positioning features for a perfusion disposable product in the tray, by providing positioning features for a sub-tray in the carrier tray, and by providing positioning features for the carrier tray in the equipment. . Features that may be used to support this alignment include reference surfaces, pins, guides, shaped surfaces (e.g., fillets and/or chamfers), springs or elastic members, etc. to facilitate registration. These may be included in carrier trays, sub-trays, equipment, perfusion disposables, or combinations thereof. For example, the present invention contemplates an embodiment in which perfusion disposables are aligned to a sub-tray, the sub-tray is aligned to a carrier tray, and the carrier tray is aligned to equipment (see FIGS. 9A and 9B). It is not intended that all of these alignments will be required, and in fact some steps of the alignment may be omitted. For example, any of the features described above may be used to align the sub-tray directly to the instrument and no carrier tray may be required for alignment purposes.

The sub-tray may optionally be designed to capture leaks originating from perfusion disposables or equipment connections. The sub-tray may optionally include one or more optical windows that may facilitate microscopic observation or inspection. This makes it possible to mount the sub-tray on a microscope or other inspection device, allowing the chips to be viewed without having to remove each disposable item from the tray. Correspondingly, the sub-tray can optionally be designed to minimize the imaging distance, for example by lying flat on or fitting into the microscope stage. The system may optionally include means for maintaining one or more perfusion disposables within the sub-tray. For example, a perfusion disposable may be clipped to a sub-tray, and the clip features may be present on the perfusion disposable, the sub-tray, an additional substrate, or a combination thereof. The system may optionally include means for retaining the sub-tray within the carrier tray. For example, a sub-tray can be clipped to a carrier tray, with clip features present on the sub-tray, the carrier tray, an additional substrate, or a combination thereof.

It may be convenient to split some desired features between the carrier tray and one or more sub-trays. For example, a sub-tray can provide an optical window and a carrier tray can be designed to capture leaks. For example, it may be desirable to include a sub-tray even when the carrier tray is designed to support only one sub-tray.

The same equipment can support different tray or sub-tray types, as well as different numbers of trays. For example, the equipment may accommodate two different tray types, and each tray type may be designed for a different type of perfusion disposable product. In this case, the tray can essentially act as an adapter to fit different perfusion-disposable types into the same equipment.

In one embodiment, the invention also provides a microscope stage, stage-insert, or adapter (e.g., plunging stage insert) designed to receive one or more chips, perfusion disposables, trays, or sub-trays. Consider. These can facilitate "dropping" numerous chips for imaging, while the chips remain securely on the stage (this avoids shaking as the microscope stage moves, for example).

C. Engagement of perfusion disposables and equipment

In one embodiment, the present invention contemplates a pressure-driven system for biological cultures in a fluidic device that applies pressure (whether positive or negative) to one or more fluid elements. These fluid members include, for example, chips, reservoirs, irrigation disposables, pressure covers, or combinations thereof. In this system, an equipment connection connects to each fluid element or elements to apply pressure as needed. This connection is typically associated with establishing a gas seal, but in some embodiments a tight seal is not necessary (e.g., pressure-regulation can maintain a desired pressure despite gas leakage). Without loss of generality, the following description is presented as establishing sealing, but is intended to also include embodiments in which sealing is not required.

In the present disclosure, systems and methods for establishing a pressure connection between biological culture equipment and one or more fluid members are contemplated. In particular, in one embodiment, the one or more fluid members are raised to contact one or more pressure manifolds included in the equipment, the one or more pressure manifolds are lowered to contact the one or more fluid members, or A system that is a combination of is considered. In some embodiments, the raising or lowering engages multiple fluid elements and equipment in unison (e.g., through a single actuation or single movement) (see FIGS. 9A and 9B), and simultaneously engages multiple microfluidic devices (e.g., herein connect one or more embodiments of the perfusion manifold assembly discussed in).

Some embodiments where the fluid member is raised include one or more platforms with one or more fluid members disposed thereon. In this embodiment, one or more platforms can be raised to effect the raising of one or more fluid members (Figure 6). In some embodiments, the equipment or system includes mechanical means (35) to manually achieve the raising or lowering associated with the erection of the pressure connection. These mechanical means 35 for manual actuation may include movement of a user-accessible control surface, which may include, for example, a level, pull/push knob, rotation control, or a combination thereof.

In some embodiments, the equipment or system includes a mechanical actuator 51 to facilitate the raising or lowering associated with the erection of the pressure connection (see FIGS. 9A and 9B). Such mechanical actuators may include, for example, one or more pneumatic components 52 (e.g., cylinders), hydraulic components (e.g., cylinders), solenoids, electric motors, magnets (e.g., mechanically fixed magnets that move into place), or combinations thereof. In some embodiments, mechanical operation may be computer controlled. In some embodiments, mechanical actuation is augmented by manual control (e.g., using any of the means for mechanical control described above), for example to provide manual retraction. A user interface on the device can control this process.

Regardless of whether operation is manual or automatic, in some embodiments, the system further includes one or more mechanisms for increasing the applied mechanical force. This may be desirable to provide sufficient force on the pressure joint to obtain a sufficient or sufficiently firm seal. These mechanisms for increasing the applied mechanical force may include levers, cams, pneumatic or hydraulic amplifiers, or combinations thereof.

In some embodiments, mechanical movement can be controlled and/or constrained using various mechanical components or designs known in the art. These mechanical components or designs include, for example, rails, guide lots, pivots, cams, four-rod connections, etc. It is important to note that the upward or downward movement can be linear, but does not have to be. For example, in some embodiments rotational actions (e.g., for pivoting) or compound actions (e.g., for linking) are desirable.

Although we have described the elevations or depressions and features present on top of the bottom of various substrates, those skilled in the art will recognize that the description can also be applied to lateral movements or movements along other axes (not necessarily linear movements); It will be understood that it may be applied to features present on any side or orientation. Additionally, although it has been suggested above that one or more fluid members are disposed below one or more pressure manifolds, one of ordinary skill in the art will recognize that the pressure manifold could instead be located below the fluid members ( It will be appreciated that, for example, the pressure connection may be disposed on the lower surface of the perfusion disposable.

This embodiment (shown in the attached drawings) includes two mechanics, each of which allows six irrigation disposables to interface with the pressure manifold 50 in a single action. In this embodiment, the pressure manifold is lowered into contact with the perfusion disposable (or optionally with a pressure cover covering the perfusion disposable) using an electrically controlled pneumatic actuator (FIG. 9B). The force of the actuator is directed using a cam system, which also increases the applied force due to its mechanical advantage. The mechanism shown is also bi-stable, meaning that if the actuator pushes the manifold up or down, the force can be released while maintaining the position of the manifold. This may help reduce fever.

D. Pressure manifold and distribution manifold

In many applications of pressure-actuated systems, it is desirable to distribute one or more pressure sources to two or more fluid members (e.g., fluid chips, irrigation disposables, reservoirs, pressure covers, or combinations thereof). For example, it may be desirable for two or more perfusion disposables to share a single set of pressure regulators to reduce the number of regulators in the system (e.g., providing a different set of regulators for each perfusion disposable). In contrast).

In one aspect of the disclosure, equipment includes one or more distribution manifolds. The distribution manifold includes one or more fluid conduits adapted to distribute one or more pressure sources to two or more fluid members (e.g., fluid chips, irrigation disposables, reservoirs, pressure covers, or combinations thereof) For example, gas channels or tubes). Correspondingly, the distribution manifold may comprise one or more pressure injection ports, which may for example be adapted to communicate with one or more pressure regulators (each injection port may communicate with a single or multiple regulators). possible). In one embodiment, the distribution manifold can also have pressure regulating components (valves, pressure sensors, pressure sources) integrated into the manifold itself. Similarly, a distribution manifold may include, for example, two or more connections that may be adapted to communicate with one or more fluid members. In some embodiments, the two or more connections include at least one region comprising an elastomeric or flexible material. Examples include gaskets, o-rings, etc. made from materials including silicone, SEBS, polypropylene, rubber, Viton, etc. These regions, including elastomeric or flexible regions, can help provide or improve fluid sealing. Such elastomeric or flexible regions may also be included in pressure manifolds other than distribution manifolds to provide similar benefits.

In addition to distributing pressure, which can be used, for example, to provide pressure-driven flow, distribution manifolds may be used for other purposes, such as mechanical compression (e.g., to actuate mechanical forces in organs on a chip). Alternatively, the pressure used to create a gas flow within the fluid member may be distributed to provide compression. Moreover, the distribution manifold can optionally distribute one or more liquids. These liquids include, for example, cleaning solutions, disinfection solutions, working liquids (e.g. for liquid-handling or flow control purposes), tissue-culture media, test agents or compounds, biological samples (e.g. blood), Or a combination thereof may be included. In some embodiments, a distribution manifold may include a working fluid, membrane, and/or plunger disposed to transmit pressure. For example, a working fluid can be used to reduce the amount of gas needed to establish a desired pressure, or to facilitate more accurate volumetric control. Membranes, plungers, and/or working fluids can be used to isolate fluids used in different parts of the distribution manifold (e.g., 5% CO2 tissue-culture on the “reservoir side” of the distribution manifold). Isolate the aircraft from dry air on the operating side).

In many applications, it is desirable to enable proper functioning of equipment even when fewer fluid members are engaged than the equipment can accommodate. For example, it is often desirable for equipment including a dispensing manifold designed to interface with six perfusion disposables to still support proper operation of the equipment when only four perfusion disposables are present. For example, it may be undesirable for gas to escape through a connection intended for a removed irrigation disposable, so that gas release may reduce the gas pressure or deplete the gas supply. These considerations are relevant even if there is no distribution manifold (i.e. even if there is a non-distribution pressure manifold).

According to one aspect of the disclosure, a pressure manifold (or specifically a distribution manifold) includes one or more devices adapted to controllably block one or more fluid (e.g., gas) conduits included in the manifold. May include valves. A variety of suitable valves are known in the art, such as pinch valves, screw valves, needle valves, ball valves, spring-loaded valves, poppet valves, umbrella valves, Belleville valves and the like. In some embodiments, one or more valves are controlled by the user. For example, the user can configure the valve to match the configuration of the perfusion disposable being used. In some embodiments, one or more valves are electronically controlled. For example, the software may configure the valve according to knowledge of the experimental setup or other information available thereto. In some embodiments, one or more valves are controlled by sensing whether an intended fluid member is present, such as to shut off a gas line when a fluid member is removed. Such sensing may be accomplished by electrical means (e.g., contact switches, circuit-closing conductors), optical means (e.g., optical gates), magnetic means (e.g., magnetic switches), or mechanical means (e.g., levers, button). In some embodiments, one or more sensing members influence one or more valves using intervening software or electronic hardware. In some embodiments, the one or more sensing members affect one or more valves directly (eg, by mechanical coupling to the valve or by electrical signaling to the valve). As a specific example, the presence of a irrigation disposable can act to press on a protruding feature, which affects the condition of the valve. This configuration is well suited for, for example, pinch valves, spring valves, poppet valves, or umbrella valves because, in some embodiments, the depressed protruding features can act directly on the valve to increase flow rate.

In some embodiments, it is desirable or convenient to include the one or more valves in one or more connections with a fluid member. This may be desirable, for example, since many successful valve designs are known that respond to forces present at the outlet. Examples of such valves include Schrader valves, Dunlop valves, Presta valves, umbrella valves, variants thereof, and related valves. As a specific example, a Schrader valve may be incorporated into the connection to the pressure shroud such that when the pressure shroud is present, this acts to press against the central stem of the Schrader valve, thereby allowing gas flow.

Valves suitable for inclusion in a connection to a fluid member as described above often have their control features located in the center of the valve (e.g., pins of a Schrader valve) (Figure 11A). However, this may present difficulties in some potential embodiments because corresponding features must be provided on the fluid member to press against this central control feature. An alternative approach is described herein. 11A and 11D, the pressure manifold 50 or distribution manifold may include a valve 59, such as a Schrader valve (or any valve described above), and add a shuttle 61. It can be included as . The shuttle includes a first surface facing a location of a potential fluid member and a second surface facing the valve. The first surface is designed to allow contact from the fluid member at a desired location. For example, the first surface may be designed to receive contact from the periphery of a port that may be present, for example, on the pressure shroud 11 ( FIG. 11D ). The second surface is designed to mechanically engage with a control surface of the valve, which may for example be at the center of the valve. A further advantage of this approach is that the thickness of the shuttle can be adjusted to control, for example, the distance from the fluid member at which the valve will open.

As further shown in FIGS. 11A and 11C, the connection may optionally be at least partially covered by an elastic, flexible or deformable substrate, such as a flexible film (e.g., a silicon film) 60. The presence of such an elastic, flexible or deformable substrate can assist in sealing the fluid member to the manifold 50. The elastic, flexible or deformable substrate may be, for example, a membrane, a gasket or a suitably shaped plug, which may include, for example, silicone, SEBS, Viton, polypropylene, rubber, PTFE, etc. As shown, an elastic, flexible or deformable substrate can be held in place by capturing it by an additional component (e.g. a cover plate 63 in this embodiment). However, elastic, flexible or deformable substrates can also be maintained in a variety of other ways, for example by bonding, attaching, welding, etc.

The desired functionality of the embodiment shown in FIGS. 9B, 11A and 11D is illustrated herein by way of example: the pressure cover 11 of the perfusion manifold assembly 10 having a ridge around the equipment connection is connected to the pressure manifold 50 come into contact with As the cover moves closer to the valve, the ridges of the cover begin to form a pressure seal against the silicone membrane of the manifold. As the cover advances, the shuttle slowly moves upward and at some point begins to press the center pin or poppet (65) of the Schrader valve (59). However, according to the example above, the shuttle will be designed so that a sufficiently good gas seal is formed before the pin of the valve is pressed sufficiently to open the Schrader valve 59. When the valve is open (ideally not before) gas can flow between the manifold (50) and the pressure cover (11). It is important to note that in this embodiment, the Schrader valve does not sense the presence of the irrigation disposable (or pressure shroud) as a single unit, but rather senses the presence of each pressure-shroud ridge independently. These embodiments may provide an additional advantage in that they can accommodate different configurations of pressure sheaths or irrigation disposables, for example, configurations that use only four of the five ports shown.

FIG. 10A shows one embodiment of a PD engagement surface 54 of a pressure manifold 50, which is a distribution manifold, and shows the elastomeric region acting as a gasket to improve gas sealing to fluid members. In this embodiment, a gas seal can be formed by compressing these elastomeric regions against ridges present on top of the pressure cover 11 disposed on the perfusion disposable product 10. The distribution manifold 50 shown provides the pressure (positive or negative pressure) used to generate pressure-driven flow in each of the six pressure shrouds, as well as the pressure used to actuate mechanical stretch within the contained on-chip organ device. (positive or negative pressure) can be dispensed (in this example, each of which is placed within a perfusion disposable, which is covered with a pressure cover). The distribution manifold shown includes several Schrader-like valves (see Figure 11D).

As the manifold engages the PD, the valve seal engages the sealing teeth or ridges on the top of the cover (see Figure 2C), forming a seal for transferring pressurized gas from the manifold to the reservoir chamber. The poppet 65 (FIG. 11D) acts as a backing to provide a hard surface for the sealing tooth on the cover to compress the valve seal. This transfers the load from the cover to the Schrader valve (59), actuating it when the PD is in position. At the same time, the Schrader valve (or similar type of valve system) operates by engaging the PD cover, allowing all gas to flow from the pressure regulator to the PD. If the PD is not in the respective position, the valve prevents any gas flow.

Spring shuttle 55 (FIG. 10B) provides a load on the cover assembly 11 to create a reservoir chamber-to-cover assembly seal (e.g., a pressure lid-to-reservoir seal) (FIG. 2D). In operation, deflection of the valve seal and displacement of the poppet 65 occur when the PD engages.

Alternatively, the lid compressor (FIG. 10C) provides a load to the cover assembly to create a reservoir chamber-cover assembly seal (e.g., a pressure lid-to-reservoir seal).

In one embodiment, each valve assembly has optional spring, flexible or elastic components built into it to allow pressure to be applied independently to each seal. In one embodiment, the spring (or similar member) is an integral part of the valve function, but may have an additional function by using it to apply pressure to a sealing tooth on the reservoir lid. A spring (or similar member) may act to restore and apply pressure to the shuttle against the fluid member, thereby providing or improving a gas seal. By applying this load independently to each seal member on the cover, a design is created that is more robust both to variations due to manufacturing tolerances and to how different PDs are loaded on the equipment.

In some embodiments, one or more described valves are controlled by software or the user. For example, a user or software may aim to block gas flow even when a fluid member (eg, perfusion disposable) is present at the corresponding connection. For example, this may be desirable if the user suspects or the software or sensor detects that there is excessive gas flow into the fluid member, perhaps because the fluid member has been damaged. The pressure manifold (whether a distribution manifold or not) may further include sensors, such as pressure sensors, flow sensors, etc.

E. Pressure and flow control

In one embodiment, a flow rate of 5 to 200 uL/hr, and more preferably 10 to 60 uL/hr, is preferred through one or more microchannels of the device. In one embodiment, this flow rate is controlled by gas pressure applied from a pressure manifold (described above). For example, when 0.5 to 1 kPa is applied, this nominal pressure produces a flow rate of 15 uL/hr to 30 uL/hr in one embodiment.

In addition to maintaining control over the gas pressure over time (and thereby maintaining control over the flow rate), in some embodiments, a device that can be applied by a process of engaging or disengaging a manifold to a perfusion disposable product. Gas pressure must also be addressed. That is, in a particular embodiment, the engagement stage of the manifold has been observed to generate a pressure surge of as much as 100 kPa for the gas present within the reservoir contained in the perfusion disposable. This can result in spikes in flow rate and/or undesirable pressure on the coupled microfluidic device. In the special case where the coupled microfluidic device includes a membrane, undesirable pressure surges can deform the membrane, cause transmembrane flow, and/or damage any contained cells.

Without wishing to be bound by theory, the mechanical force applied to the pressure cover by the manifold deforms one or more compliant materials contained in the pressure cover or irrigation disposable (e.g., compresses any gaskets, etc.), The pressure surges described may occur. This strain can act to reduce the volume of gas present in the reservoir and increase its pressure. The opposite effect, leading to a negative pressure surge, can occur during manifold separation; Those of ordinary skill in the art will understand that this discussion primarily considers the positive pressure surges typical of manifold engagement, but may similarly apply to negative pressure surges that may be typical during manifold disengagement. Whether at positive or negative pressure, if the gas volume in the reservoir is small (this can happen if the liquid volume in the reservoir is high, for example in a preferred embodiment the volume is greater than 3 milliliters, especially greater than 5 milliliters) when) surges can be particularly problematic. These engagement surges may take time to dissipate because typically excess pressure must be vented. In embodiments where the pressure shroud includes a strainer, this strainer may provide dominant resistance to exhaust, which dictates the dynamics of pressure surge dissipation. In one embodiment, the present invention contemplates reducing the exhaust resistance in the system to avoid such surges, reduce the magnitude of the surge and/or reduce the duration of the surge. In one embodiment, the present invention contemplates selecting a strainer to alleviate pressure surges during cartridge insertion and removal.

In this regard, reference is made to Figure 2. 2A shows one embodiment of a cover assembly 11 comprising a cover or shroud having a strainer 38 and a plurality of ports (e.g., through-hole ports) associated with corresponding holes 39 in the gasket. This is an exploded view of . Figure 2B shows the same embodiment of the cover assembly with the strainer 38 and gasket located within (and below) the cover. In one embodiment, the strainer for the discharge pressure port is selected for low gas-flow resistance. For example, to reduce resistance and quickly dissipate manifold-engaging associated gas pressure (discussed above) to avoid prolonged spikes in flow rate, some embodiments use a 0.2 micrometer filter (used at the injection pressure port). Instead, use a 25 micrometer filter. In a particular embodiment, a filter with an average pore size of 25 um (filter 4901, commercially available from Forex) does not compromise sterility at 1/8 inch thickness. These filters maintain sterility despite their large pore size (much larger than typical bacteria/spores) by creating complex passages through their thickness which are significantly thicker compared to previously mentioned filter membranes/sheets.

It is important to note that the design of the injection and discharge pressure ports may require different treatment with respect to exhaust resistance. For example, in embodiments where the perfusion disposable or microfluidic device includes a resistor, the pressure applied to the resistor side (whether the resistor is installed upstream or downstream of the region of interest) is typically applied to the region of interest (e.g., containing cells). It does not act directly on (can). This may be the case, for example, when liquid flow through a resistor causes a pressure drop. Conversely, because there may not be enough pressure drop to provide any degree of isolation, pressure surges on the non-resistor side (whether inlet or outlet) may act directly against the area of interest. In the particular example where there is a resistor on the inlet side of the region of interest, a pressure surge over the inlet may produce a corresponding surge in flow rate, but minimal increase in pressure is experienced inside the region of interest; Conversely, a pressure surge at the outlet can produce both a surge in flow rate and a surge in experienced pressure. In some applications, for example when the microfluidic device includes a membrane, pressure in the region of interest can be significantly more detrimental than transient spikes in flow rate. Therefore, in this embodiment it may be advisable to include only low-resistance filters at the discharge port and more traditional (higher resistance) filters at the injection port, as these may provide advantages for flow control ( discussed further in this disclosure).

Having discussed the problem of engagement/separation surges, we now turn to the problem of control of gas pressure, especially in the low pressure range. Some commercially available pressure regulators (or pressure controllers) advertise an adjustable pressure range with a lower pressure limit that is above zero. For example, SMC ITV-0011 regulators are marketed for pressure control in the 1 to 100 kPa range (their linearity has been observed to be poor in the 0 to 1 kPa range). In some applications, it may nevertheless be desirable to achieve flow rates corresponding to pressures lower than the specified or linear range of commercially available regulators. Moreover, the accuracy of commercially available pressure regulators is typically a percentage of “full scale,” implying that control at the lower limit of pressure is characterized by a greater percentage of variability. In some applications, this may translate into low accuracy or fidelity in pressure control to the lower end of the usable range. In one embodiment, one or both of these challenges are addressed by a form of “pulse-width modulation” incorporated into the pressure actuation method.

In this regard, reference is made to Figure 6. In one embodiment, culture module 30 includes a removable tray 32 for positioning the assembly-chip combination, a pressure surface 33, and a pressure controller 34. In one embodiment, the tray 32 is placed in the culture module 30 and moved upward through the tray mechanism 35 to engage the tray 32 with the pressure surface 33 of the culture module, i.e. the perfusion manifold. The cover or sheath (11) of the assembly engages the pressure surface of the culture module. Rather than the pressure controllers being “on” all the time, they are switched in an “on” and “off” pattern (or between two or more settings). Accordingly, the switching pattern can be selected such that the average value of the liquid operating pressure in one or more reservoirs corresponds to the desired value. This approach is similar to pulse-width modulation (PWM), pulse-density modulation (PDM), delta-sigma modulation (DSM) techniques and similar techniques known in the field of electrical engineering. In the case of pulse-width modulation, for example, a regular switching period is selected. Within each period the pressure controller can be turned on for the set pressure for the desired duration or turned off for the remainder of the switching period. The longer the switch-on time is than the switch-off time, the higher the overall average pressure applied. The term “duty cycle” describes the ratio of “on” time to the switching period; Low duty cycle corresponds to low pressure because the pressure is off most of the time. Duty cycle is expressed as a percentage, with 100% being completely “on”. By using this type of “pulse-width modulation” with a pressure controller, it has been shown that the average gas pressure can be reliably maintained below 1 kPa using a regulator that does not provide linear control in that range. In a particular embodiment, the pressure regulator is used in its typical “linear” manner for pressures from 1 kPa to 100 kPa, with an “on pressure” of 2 kPa and an “off pressure” of 0 kPa for a mean-pressure setpoint of 0 kPa to 1 kPa. It switches to pulse-width modulation using “pressure”. In another example, pulse-width, pulse-density or delta-sigma modulation can be used to control the average pressure between 0.3 and 0.8 kPa.

Although the disclosed method may involve applying a pulsatile pressure pattern to the pressure envelope, it has been experimentally confirmed that the strainer helps smooth the pressure exerted on the liquid by the reservoir. Without wishing to be bound by theory, the degree of smoothing increases with the resistance of the filter to gas flow and the volume of gas in the reservoir (typically decreasing as more liquid is present). Similarly, analogies to electrical circuits show that shorter switching periods increase smoothing. Accordingly, a person skilled in the art can select the degree of smoothing by selecting the resistance of the gas filter, setting a lower limit on the gas volume, and selecting the switching period or modulation pattern. It is important to ensure that the pressure regulator controllably regulates the pressure at a rate sufficient to reproduce the designed pressure modulation pattern. In some embodiments, a 0.2 um filter (Porex filter membrane) and a switching period of 10 seconds provide desirable smoothing. In other embodiments, a 0.4 um filter may be used.

Detailed Description of Preferred Embodiments

A. Drop-to-drop connection

A drop-to-drop connection is contemplated as an embodiment for installing a microfluidic device in fluid communication with another microfluidic device, such as, but not limited to, installing a microfluidic device in fluid communication with a perfusion manifold assembly. . When the devices are placed in fluid communication with each other, a bubble 40 may form as shown in FIGS. 14A and 14B, where the first surface 87, including the first fluid port 89, is connected to the second surface ( 88) and aligned along the second fluid port 90. In one embodiment, a drop-to-drop connection is used to reduce the chance of bubbles being trapped during connection. Air bubbles are particularly challenging in microfluidic geometries because they are pinned to the surface and difficult to flush out with fluid flow alone. They pose additional challenges in cell culture devices because they can damage cells through a variety of means.

In one embodiment, droplets are formed on the surface of the device in areas around and above the fluid path or port, as shown in FIGS. 15A, 16A, 16B, 16D and 17-21. When the surfaces come close to each other during connection, the drip surfaces are connected without introducing any air bubbles. In practice, it is difficult to maintain the alignment and stability of the drop during manual device manipulation. Additionally, in situations where the binding number is high, liquid tends to drain from the device quickly and in an unstable manner. Here, a number of solutions are described to address both the problem of maintaining a stable drop on a device surface and the problem of guiding drop-to-drop engagement of two primed devices in a controlled and robust manner.

FIG. 16A shows one embodiment of contacting a microfluidic device with a fluid source or another microfluidic device, wherein the microfluidic device is approached from the side and a side track engages a portion configured to fit into the side track. FIG. 16B shows one embodiment of contacting a microfluidic device with a fluid source or another microfluidic device, wherein the microfluidic device is approached from the side and down the side so that a side track engages a portion configured to fit into the side track. wherein the side tracks include an initial linear portion and a subsequent angled portion, causing both lateral and upward movement of the microfluidic device as the microfluidic device moves in engagement with the side tracks, thereby establishing drop-to-fluidic communication. A point connection is created (Figure 16c). 16D shows another approach to contacting a microfluidic device with a fluid source or another microfluidic device, wherein the microfluidic device pivots at a hinge, joint, socket, or other pivot point on the fluid source or other microfluidic device. (arrows show general direction of movement).

17 is a schematic diagram showing a defined droplet 22 on the surface 21 of a microfluidic device 16 in a pathway or port, where the droplet covers the entrance of the port and protrudes above the port, which is connected to the microchannel and communicate fluidly.

18 is a schematic diagram showing a defined droplet 22 on the surface 21 of a microfluidic device 16 in the region of a path or port, where the droplet is on a molded pedestal or mount 42 and in the region of the port. It covers the inlet and protrudes above the port, which is in fluid communication with the microchannel.

19 is a schematic diagram showing a defined droplet 22 on the surface 21 of the microfluidic device 16 in the region of a passage or port, where the droplet is on a gasket 43 and covers the inlet of the port. , protrudes above the port, and the port is in fluid communication with the microchannel.

20 is a schematic diagram showing a defined droplet 22 in which a portion of the droplet in the region of the path or port is located below the surface 21 of the microfluidic device 16, where the droplet is a shaped depression or depression ( 44) and covers the entrance of the port, a portion of which protrudes above the surface, and the port is in fluid communication with the microchannel.

FIG. 21 is a schematic diagram showing a defined droplet 22 with a portion of the droplet located below the surface 21 of the microfluidic device 16 in the region of the path or port, where the droplet is within the surrounding gasket and at the inlet of the port. covers, and a portion protrudes above the gasket.

FIG. 22 is a schematic diagram illustrating a surface modification embodiment that uses a sticker to define a droplet on the surface of a microfluidic device 16 at a port, where the port is in fluid communication with a microchannel. Figure 22A utilizes a hydrophilic adhesive layer or sticker 45, with droplets 22 spreading from the edge of the sticker and bound by the surrounding hydrophobic surface. Figure 22b shows a droplet 22 spread over the hydrophilic surface of the device and bound by a surrounding hydrophobic surface 45 created by one or more adhesive layers or stickers on each side of the port, wherein the port Fluid communication with microchannels.

23 is a schematic diagram illustrating a surface modification embodiment, utilizing a surface treatment (e.g., chemical vapor deposition, plasma oxidation, Corona, etc. - indicated by an arrow drawn downward) in conjunction with a mask 41. ; In one embodiment, the microfluidic device 16 is made of a naturally hydrophobic material that becomes hydrophilic in the absence of the mask but remains hydrophobic in the presence of the mask upon such surface treatment. After surface treatment, the mask can be removed and the channels can be filled with fluid to create droplets that protrude above the surface but are confined by regions that remain hydrophobic (see Figure 17).

Figure 24 is a schematic diagram of one embodiment of a drop-to-drop connection scheme that uses a combination of geometry and surface treatment to control droplets. FIG. 24A shows an embodiment of a microfluidic device or “chip” comprising fluidic channels and ports, with a raised area (e.g., a pedestal or gasket) at each port. If other parts of the device (i.e., parts other than the pedestal or gasket) are treated (e.g., plasma treated) to render them hydrophilic, the naturally hydrophobic pedestal or gasket must be masked (e.g., in Figure 24a) during the plasma treatment. It can be prevented from becoming hydrophilic by protecting it with a member 41 (shown on a stand or gasket). After plasma treatment, the mask is removed (e.g., peeled from the surface of the base or gasket). FIG. 24B shows a fluid-filled hydrophilic channel with the droplet radius balanced at each end (i.e., at the port opening), and the droplets 22 are confined by a hydrophobic gasket surface. 24C shows an upwardly protruding droplet 22 approaching (but not yet touching) a portion of the mating surface of a flow-through manifold assembly with a protruding droplet (in this case, droplet 23 protruding downwards). A portion of the microfluidic device of FIG. 24B is shown with . FIG. 24D shows the same portion of the microfluidic device of FIG. 24C with the upwardly protruding droplet 22 of the microfluidic device in contact (fused) with the downwardly protruding droplet 23 of the perfusion manifold assembly. The droplets coalesce in a controlled manner when they are on a hydrophilic surface but are constrained by a hydrophobic surface. As previously mentioned, embodiments in which the microfluidic device (with the droplet projecting downward) is accessed above the perfusion manifold assembly (with the droplet projecting upward) are also contemplated.

Figure 25 shows an embodiment of drop-to-drop connected using only surface treatment (i.e., without geometry, such as a pedestal or gasket). Figure 25A shows an embodiment of a perfusion manifold assembly including fluid channels and ports. If other parts of the naturally hydrophobic bonding surface (i.e., other than the area surrounding the port) are treated (e.g., plasma treated) to become hydrophilic, the area surrounding the port may be masked during the plasma treatment (see Figure 25a). Protected by a member (shown as 41) covering the port and a small area of the bonding surface around the port to prevent it from becoming hydrophilic. After plasma treatment, the mask is removed (e.g., peeled from the bonding surface around the port). Figure 25B shows a hydrophilic channel filled with fluid to a given level (e.g., height of the fluid column). In some embodiments, the formed droplets can resist the pressure (gravitational head) exerted by the fluid volume. This is advantageous because it can enable drop-to-drop connections while minimizing dripping of the upper drop and stabilizing its size. Without being bound by theory, a drop resists pressure because the pressure exerted by the fluid volume is balanced by the surface tension of the drop; This surface tension is determined in part by the drop radius, which can be controlled using the designs and methods disclosed herein; For example, when a droplet is constrained by a hydrophobic region around a port, the radius of its surface is similarly constrained.

Figure 26 is a chart showing (without being bound by theory) the relationship between port diameter (in millimeters) and the maximum static head (in millimeters) that a stabilized drop can support, assuming that the fluid has the same surface tension as water. (The above model does not include the reservoir meniscus). This allows working with a variety of port diameters, providing significant process range and tolerance for user operation by selecting one that can support a substantial volume of water column in the channel (usually capable of supporting a substantial back pressure). It shows that it can be done. In yet another embodiment, the desired size of raised or protruding droplet is achieved by adjusting the pressure on the fluid.

The present invention is not intended to be limited to any particular method of controlling droplet size, orientation or direction. In one embodiment, the present invention contemplates using (or making) a surface engineered to form stable droplets. These surfaces may be hydrophilic or hydrophobic in nature, or may be treated to become hydrophilic or hydrophobic. The present invention is not intended to be limited to any one technology. However, among various methods of hydrophilic treatment (e.g., low pressure oxygen plasma treatment, corona treatment, etc.), clean technology is preferred for processing poly(dimethylsiloxane) (PDMS) microfluidic devices. In one embodiment, the present invention contemplates that a hydrophilic surface can be created (over what would normally be a hydrophobic material) using an atmospheric RF plasma. See Hong et al., "Hydrophilic Surface Modification of PDMS Using Atmospheric RF Plasma," Journal of Physics: Conference Series 34 (2006) 656-661 (Institute of Physics Publishing). In one embodiment, a mask 41 as shown in Figure 23 is used with this plasma treatment. For example, a mask can be attached to areas of the surface of the microfluidic device 16 (e.g., made of PDMS or another polymer) prior to plasma treatment to prevent these areas from becoming hydrophilic. controls which part of the PDMS chip becomes hydrophobic and which part remains hydrophobic). After plasma treatment, the mask 41 can be removed (FIG. 24) (typically by simply peeling the mask from the surface). In yet another embodiment, the present invention contemplates using plasma surface treatment in a fluorinated environment to increase the hydrophobicity of the surface. Avram et al., “Plasma Surface Modification for Selective Hydrophobic Control,” Romanian J. Information Science and Technology , Vol. 11, Number 4, 2008, 409-422.

Alternatively, these surfaces can have geometric features or shapes that allow the droplets to form or behave in a desired manner. For example, as shown in FIG. 18 the bonding surface may have a protrusion, platform or pedestal 42 with a geometry that allows the droplet to have special dimensions. As shown in Figure 19, the surface can also be covered with a structure surrounding the port through which the droplet protrudes, such as a gasket 43 or other mechanical seal, which forms two bonding surfaces (i.e., one surface from a microfluidic device). and one surface from the irrigation assembly) to prevent leakage while under compression.

Alternatively (FIG. 20), a portion of the droplet may be located in a depression or depression 44 such that a portion of the droplet is below the bonding surface 21 of the microfluidic device, as shown in FIGS. 20 and 21. You can. In yet another embodiment, an adhesive patch or sticker 45 can be placed on the surface, as shown in FIGS. 22A and 22B, to create hydrophilic or hydrophobic regions on the bonding surface of the microfluidic device.

In yet another embodiment, a combination of geometric features and surface treatments may be applied. For example, hydrophobic scaffolds or gaskets can be used (or manufactured) to allow for smaller droplet sizes. Most elastomeric polymers used in the manufacture of gaskets are hydrophobic. Such gaskets are manufactured by, for example, Stockwell Elastomerics, Inc. (Stockwell Elastomerics, Inc., Philadelphia, PA). Meanwhile, M&P Sealing offers high-quality products made from materials such as polytetrafluoroethylene (“PTFE”), perfluoroalkoxy (“PFA”), or fluorinated ethylene (“FEP”), e.g. Machine soft hydrophobic gaskets (Orange, TX, USA). These are also considered in some embodiments. If other parts of the device (i.e., parts other than the pedestal or gasket) are treated (e.g., plasma treated) to make them hydrophilic, the naturally hydrophobic pedestals or gaskets must be protected with a mask during the plasma treatment to make them hydrophilic. You can prevent it from happening.

In one embodiment, the wall of the port (or at least a portion thereof extending to the bonding surface of the microfluidic device) is or becomes hydrophilic. In one embodiment, the wall of the corresponding port (or at least a portion thereof extending to the mating surface of the perfusion assembly) is or becomes hydrophilic. In one embodiment, both the walls of the ports of the microfluidic device and the corresponding ports (or portions thereof) of the perfusion assembly are hydrophilic or become hydrophilic.

In one embodiment, the present invention contemplates a surface designed to hold droplets that resist the weight of the liquid in the reservoir (as shown in Figure 25). This is particularly important in practice because it allows a drop placed on the upper device to be easily created (i.e. when the first device approaches the second device from above). This embodiment makes it possible to simply place a measured amount of liquid into the reservoir (e.g. 100uL, 75uL, 50uL or some other amount), allowing the liquid to flow into the port, forming a droplet and stopping on its own. Importantly, this embodiment is not intended to be limited to any particular amount of liquid; in fact, accurately measured amounts of liquid are not required. To form a drop by this method, it is sufficient to aim for a specific amount as long as it is below a certain threshold (where the weight of the water overwhelms the surface tension of the drop and destroys it). It may be more or less convex depending on how much liquid is pushed above it, but the spatial extent of the drop should be the same.

The present invention is not intended to be limited to only one approach to drop-to-drop connection of microfluidic devices. In one embodiment, as shown in Figure 15A, the first microfluidic device is used to mimic one or more functions of cells in an organ of the body (i.e., to mimic one or more functions of a cell in an organ of the body, such as cell-cell interaction). While the organ microfluidic device on the chip containing the cells (mimicking action, cytokine expression, etc.) has a droplet that projects upward, the corresponding droplet on the second microfluidic device projects downward. In another embodiment, the first microfluidic device, such as an organ microfluidic device on a chip comprising cells that mimic cells in an organ in the body or mimic at least one function of an organ, has a droplet that protrudes downward. Then, the corresponding droplet on the second microfluidic device protrudes upward.

Aside from arguments about momentum, gravity alone plays a role in stable droplet formation. For example, a chip lying flat on a table experiences no significant force due to gravity. For example, if the device is tilted as part of the engagement procedure, fluid will flow from a higher point to a lower point. Accordingly, the orientation of the device, such as having the device face upward or downward in a path, may be considered another way to help constrain the droplet.

An additional aspect that controls drop volume is the fluid resistance of the device channels. For example, if a device has small channels, the fluid resistance will be high enough to maintain a nearly constant droplet volume over time, despite the presence of a force (e.g., gravity or capillary force) directing fluid flow from the device. You can. This is also true for high binding numbers. Adjustment of fluid resistance can be used as a single method of "defining the drop" or in combination with other methods such as controlling the liquid pinning geometry or controlling the wetting properties of the surface; While fluid resistance is used to control drop volume, control of the wetting properties of the surface will help control drop placement.

B. Microfluidic device

The present invention is not intended to be limited by the nature of the microfluidic device. However, preferred microfluidic devices are described in U.S. Patent No. 8,647,861 (incorporated herein by reference), which capture living cells in a microchannel, e.g., cells on the membrane of the microchannel exposed to culture fluid at a predetermined flow rate. It is a microfluidic “organ on a chip” device that includes: The surface and/or membrane of the microchannel can be coated with cell adhesion molecules to support the attachment of cells and promote their organization into tissues. If a membrane is used, the tissue may be formed on the top surface, the bottom surface, or both. In one embodiment, different cells live on the upper and lower surfaces, thereby creating one or more tissue-tissue junctions separated by a membrane. Membranes may be porous, flexible, elastic, or combinations thereof, with pores sufficiently large to allow only the exchange of gases and small chemicals, or pores sufficiently large to allow movement and passage of living whole cells as well as large proteins. In one embodiment, the membrane selectively expands and contracts in response to pressure or mechanical force, which can further physiologically stimulate the mechanical forces of the living tissue-tissue interface.

Figure 33 shows a schematic diagram of an exemplary microfluidic device or “organ on a chip” device. The assembled device is schematically shown in Figure 33A and includes multiple ports. Figure 33b shows an exploded view of the device of Figure 33a, showing a lower piece 97 with channels 98 in a parallel configuration, and an upper piece 99 with a plurality of ports 2 and a tissue-tissue interface. The stimulation zone comprises a membrane 101 between the upper piece 99 and the lower piece 97, where cell behavior and/or passage of gases, chemicals, molecules, particles and cells is monitored. In one embodiment, the injection fluid port and the discharge fluid port are in communication with the first central microchannel such that fluid dynamically flows from the injection fluid port to the discharge fluid port through the first central microchannel independently of the second central microchannel. Allow it to move. Additionally, it is contemplated that fluid passage between the injection and discharge fluid ports may be shared between the central microchannels. In either embodiment, the characteristics of the fluid flow through the first central microchannel, such as flow rate, are controllable independently of the fluid flow characteristics through the second central microchannel, and vice versa.

34 is a schematic diagram showing an embodiment having two membranes 101 and 102 with cells 103 inside the device in the first channel, in contact with fluidic channels 104 and 105, where the arrows indicate flowing Shows the direction. The three channel device allows tracking the movement or movement of cells, such as lymphoid cells, vascular cells, nerve cells, etc. In one embodiment, the membrane 101 is coated with lymphoid endothelium on its upper surface and stromal cells on its lower surface, and a second porous membrane 102 also coated with stromal cells on its upper surface and its lower surface. It is coated with vascular endothelium on its surface. The movement of these blood vessels and stromal cells can be monitored. Alternatively, a third type of cell can be placed in the center 103 and movement through the membrane can be monitored (e.g., by imaging or by detection of cells in the channel or channel fluid). The membrane may be porous or may have grooves that allow cells to pass through the membrane.

In one embodiment, the three channel device is used to measure cellular behavior of cancer cells. Tumor cells are installed in a central microchannel surrounded on the top and bottom by, for example, a layer of stromal cells on the surfaces of the top and bottom membranes. Fluid, such as cell culture medium or blood, enters the vascular channel. Fluid, such as cell culture medium or lymph, enters the lymphatic channel. This configuration allows researchers to mimic and study tumor growth and invasion into blood vessels and lymphatic vessels during cancer metastasis. The membrane may be porous or may have grooves that allow cells to pass through the membrane.

C. Seeding the device with cells

In various embodiments described above, the microfluidic chip or other device includes cells. In some embodiments, cells are seeded directly on the chip. However, in other embodiments, the chip is contained within a carrier, and the carrier is mounted on a stand to facilitate cell seeding. Figures 35A-C show one embodiment of a “seeding guide” and stand. In one embodiment, a seeding guide engages a carrier containing a microfluidic chip and holds the chip straight (e.g., for upper channel seeding) at various stages of seeding and/or coating (e.g., ECM coating). Supported upward and upside down (e.g. for lower channel seeding), improves aseptic technique. 35A shows one embodiment of the stand 100 assembled, i.e., with the two end caps 106, 107 engaged with the side panels 108, 109. Figure 35b shows the chip 16 and the carrier 17 engaged by a seeding guide, with the seeding guide approaching the stand 100. Figure 35c shows six carriers 17 with chips, each engaged with a seeding guide mounted on a stand 100. The seeding guide is adapted to receive the chip carrier (eg, in a manner similar to how a skirt engages a chip carrier); After coating and/or seeding, the same chip carrier may be connected to the flow-through manifold assembly (after being separated from the seeding guide). The seeding guide is designed to support the chip (either straight up or upside down) so that the port does not contact the table top or any other surface. This is to avoid contaminating the chip through such contact. Additionally, a seeding guide or holder may facilitate access to the chip via a pipette and/or needle and optionally utilize guide features to assist insertion into the chip port.

In one embodiment, the invention provides a stand having a) a portion configured to receive i) a chip at least partially contained in a carrier, ii) cells, iii) a seeding guide and iv) at least one seeding guide in a stable mounting position. providing steps; b) engaging the seeding guide with the carrier, thereby creating an engaged seeding guide; c) mounting the engaged seeding guide on the stand, and d) seeding the cells (e.g., using a pipette tip) while the seeding guide (together with the carrier and chip) is in a stable mounting position. Consider a seeding method, comprising seeding a chip. In one embodiment, a microfluidic device or chip includes an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device or chip comprises cells on the membrane and/or in (or on) one or more channels after seeding in step c) (e.g., the upper channel is seeded). In one embodiment of this method, multiple seeding guides are mounted on a stand and allow multiple chips to be seeded with cells. The guides must a) keep the chip surface sterile during handling, b) properly guide the pipette tip into the port during seeding, and c) clearly label the channels of the chip (e.g., upper and lower channels). d) allows loading of liquid in the channels onto the chip (as well as loading of already seeded or ECM-functionalized cells onto the chip). The stand also a) keeps the chips level so that the cells are evenly distributed across the membrane, b) allows the guide to be flipped upside down for seeding of the lower channels, and c) allows the user to hold multiple seeded chips at once. It has numerous functions, including enabling storage and storage. Accordingly, in one embodiment, after seeding in step c), the method continues by turning the chip upside down and seeding the lower channels.

Experiment

Example 1

Conditions for bonding the capping layer (Figure 2, element 13) to the backplane 14 were tested. The extruded SEBS sheet was bonded to the hot embossed plate. SEBS sheet is designed to act as a capping layer for channels formed in the COP through a high-temperature embossing process and as a fluid and gas gasket for bonded components. Testing confirmed that 1 mm thick SEBS provided a better fluid seal between the reservoir and the backplane. High temperature embossed plates were made from Zeonor 1420R. The SEBS materials used are:

A. Thickness: 1mm, Material: Kraton G1643, Mfg Process: Extrusion

B. Thickness: 0.2mm, Material: Kraton G1643 +5% polypropylene, Mfg Process: Extrusion

An oven process was used compared to a laminator. The laminator had limitations as it was not properly bonded. However, the oven process showed:

In some embodiments, the fluid layer is sealed with a film. The film may be a polymeric, metallic, biological film, or a combination thereof (e.g., a laminate of multiple materials). Examples of materials include polypropylene, SEBS, COP, PET, PMMA, aluminum, etc. Specifically, the film may be elastomeric. The film can be secured to the fluid layer by adhesives, thermal lamination, laser welding, clamping, and other methods known in the art. Films may further be used to secure and potentially fluidly connect additional components to the fluid layer. For example, a film can be used to attach one or more reservoirs to a fluid layer. In an exemplary embodiment, the film is a thermally laminated film comprising EVA or EMA. In an exemplary embodiment, the film may first be laminated to the fluid layer using a heat treatment, and then a second heat treatment may be used to attach one or more reservoirs to the fluid layer. In a different embodiment, the film includes SEBS, which is known to be capable of bonding to a variety of materials including polystyrene, COP, polypropylene, etc. by heat treatment or with the aid of one or more solvents. In this embodiment, a SEBS film can be laminated to a fluid layer (using a heat treatment or with the aid of a solvent) and a second treatment can be used to bond one or more reservoirs to the fluid layer. There are several potential advantages to using films that are elastomeric, deformable or flexible, or films that reflow during the bonding process. These advantages include, for example: manufacturing tolerances (e.g., for flatness or flatness of the manufactured part), potentially conforming to fluid layers or other bonded components (e.g., reservoirs); It relieves pressure, potentially simplifies the parallelism or alignment required during bonding (e.g., the film can be deformed to absorb errors in parallelism), and acts as a gasket to seal, for example, fluid backplanes and reservoirs. creates a fluid seal between them. SEBS is particularly advantageous as a bonding film because it can bond under moderate temperatures (typically below 100 C) without significant reflow. Reflow can be undesirable because it runs the risk of filling and clogging the fluid channels. Because there is no significant reflow, SEBS can better maintain the dimensions and structure of the fluid channels and other features of the fluid layer compared to reflow materials (e.g., typical thermally laminated films). In different embodiments, film thickness may range from 10 um to 5 mm. The film may include various fluid ports or channels. The film need not be flat and can have a variety of three-dimensional shapes.

Example 2

In this example, one embodiment of a protocol for chip activation is discussed. This example assumes that all work is performed under a hood using aseptic techniques and that all work areas are sterile (or made sterile).

Part I: Fabrication of Chips

A. Spray 70% ethanol on the outside of the chip package and wipe it before putting it in the hood.

B. Open the package inside the hood and take out the chips in the chip carrier (take them out together).

C. Place the chip in the chip carrier into a large sterile dish.

i. Chip carriers are treated only as their wings. Always use tongs to handle chips. Connect the chip surface to the cell culture area. Avoid contact with the chip surface with your hands, and hold the chip unit until it is flat.

D. Allow the vial of Emulate Reagent 1 (ER1) powder (containing cross-linker) to fully equilibrate to ambient temperature before opening to prevent condensation in the storage container - ER1 is sensitive to moisture and light.

E. Turn off the biosafety hood light.

F. Reconstitute the powder with Reagent 2.

i. Add 1 ml of Emulate Reagent 2 (ER2) (with buffer) directly to the ER1 storage container and mix thoroughly by inverting 3 times.

ii. Cover the ER1 solution with tin foil to prevent deterioration by light.

G. Wash the chip.

i. The chips are oriented horizontally within the hood.

ii. Pipette 100 ul of ER2 solution using the tip.

iii. Place the pipette in a fully vertical position and insert it into the lower channel - if the port is difficult to find, feel the surface near the port to explore it.

iv. After locating the port, insert the tip into the port (connect it securely).

v. Flush with 100 ul of ER2 solution and continue to press the pipette plunger (if you see fluid coming out, the flush was successful; if you see fluid coming out of the same injection port, the tip is not injected properly and follow step iv (repeat).

vi. To remove the tip, use sterile forceps to gently press the chip body to remove the tip, while continuing to depress the pipette plunger.

vii. Suction the outlet flow.

viii. Repeat the same procedure for cleaning the upper channel.

ix. After cleaning, the upper channel is first emptied by aspirator, followed by the lower channel.

H. Inject ER1 solution into both channels.

i. Pipette 30 ul of ER1 solution using the tip.

ii. Navigate the injection port of the lower channel using a pipette tip on the top of the chip surface near the port.

iii. After locating the port, insert the tip into the port (connect it securely).

iv. Inject 30 ul of ER1 solution and continue to depress the pipette plunger (if you see fluid coming out, the injection was successful; if you see fluid coming out of the same injection port, the tip is not injected properly and follow step ii (repeat).

v. To remove the tip, use sterile forceps to gently press the chip body to remove the tip, while continuing to depress the pipette plunger.

vi. Aspirate excess fluid from the chip surface (avoid contact with the ports).

vii. Repeat the same procedure for the upper channel using 50 ul of ER1 solution.

viii. Avoid introducing bubbles. Inspect the channel under a microscope to ensure that no bubbles are present; if bubbles are present, re-inject the ER1 solution.

I. Place the chip directly under the UV lamp, ensure that there is a UV light device in the hood and the light is turned on, and use the button to adjust the settings back to the "constant" state.

J. Treat with UV light for 20 minutes.

K. After UV treatment, gently aspirate ER1 from the channel through the same port until there is no solution in the channel.

L. Wash both channels with 100 ul of ER2 solution, then with 200 ul of dPBS.

Part II: Coating

A. Prepare the ECM according to the manufacturer's instructions. If the manufacturer directs, it is recommended to aliquot and freeze the ECM. Avoid multiple freeze-thaw cycles.

B. Calculate the total volume of ECM solution.

Minimum volume for channel

i. Top: 50ul

ii. Bottom: 20ul

iii. ECM diluent: customized for ECM, prepared on ice.

If using Matrigel, refer to the Matrigel protocol (the Matrigel protocol ensures that you have a “slush” of ice, do not touch it, and any warming will destroy the Matrigel).

C. Aspirate dPBS from the channel.

D. Load the channel with ECM solution.

i. Pipette 30 ul of cold ECM solution using the tip.

ii. Navigate the injection port of the lower channel using a pipette tip on the top of the chip surface near the port.

iii. After locating the port, insert the tip perpendicularly into the port (connect tightly).

iv. Inject 30 ul of ECM solution and continue to depress the pipette plunger (if you see fluid coming out, the injection was successful; if you see fluid coming out of the same injection port, the tip is not injected properly and follow step ii (repeat).

v. To remove the tip, gently press the chip body using sterile forceps to remove the tip.

vi. Aspirate excess fluid from the chip surface (avoid contact with the ports).

vii. Repeat the same procedure for the upper channel using 50 ul of ECM solution.

E. Incubate at 4°C overnight or at 37°C for 2 hours.

F. Seal the dish containing the coated chips using parafilm.

Example 3

This example provides one embodiment of a protocol for seeding cells inside a chip in the upper channel (which is horizontally oriented unless otherwise indicated). This example assumes aseptic technique and a sterile environment.

It should be noted that although some cells require very specific seeding conditions, generally optimal seeding density is achieved when cells are closely spaced in a planar monolayer. From this separation, most primary cells will attach and spread into the confluent monolayer.

See “Gravity Washing” below. This means a) placing a drop (bolus) of medium (100uL) onto the port on one side of the channel, making sure no air bubbles are introduced within the port itself, and b) flowing it through the chip, always leaving the outlet port. It involves aspirating excess medium from.

A. Move the chip to the hood.

B. Place it in a sterile dish (e.g., 15 mm culture dish).

C. Lightly wash the chip.

i. Pipette 200 ul of cell culture medium using the tip.

ii. Navigate the injection port of the lower channel using a pipette tip on the top of the chip surface near the port.

iii. After locating the port, insert the tip perpendicularly into the port (connect tightly).

iv. Flush 200 ul of media and continue to press the pipette plunger (if you see fluid coming out, the flush was successful; if you see fluid coming out of the same injection port, the tip is not injected properly and proceed to step iv. repeat).

v. To remove the tip, use sterile forceps to gently press the chip body to remove the tip, while continuing to depress the pipette plunger.

vi. Aspirate the drained fluid.

vii. Repeat the same procedure for cleaning the upper channel.

viii. Repeat the wash steps one more time for both channels.

ix. Add medium dropwise to the injection and discharge ports (100 ul each).

D. Cover the dish and place it in the incubator until the cells are ready.

E. Prepare cell suspension and count cells.

F. Seeding density is specific to upper and lower channels, cell type, and customized needs.

i. Upper channel: e.g. Caco2 cells: 2.5 million cells/ml

ii. Lower channel: e.g. HUVEC: full growth

G. After counting the cells, adjust the cell suspension to an appropriate density.

H. For upper channel seeding, place the dish containing the chips in the hood and aspirate off excess medium on the chip surface (handle chip carriers only by their wings, keep chip carriers flat, do not pick up! This will ensure uniform distribution of cells across the chip culture membrane).

I. Gently aspirate the cell suspension before seeding each chip.

J. Pipette 50 μl of cell suspension and seed into the upper channel (when the chip is in a horizontal position, the upper channel is the lower right port) (use 1 chip first).

i. Place the pipette in a fully vertical position and insert it into the upper channel (vertical ensures smooth introduction into the chip and more even cell distribution).

ii. Inject 50 ul of cell suspension and continue to depress the pipette plunger (if you see fluid coming out, the injection was successful; if you see fluid coming out of the same injection port, the tip is not injected properly and see step ii (repeat).

iii. To remove the pipette, gently press the chip body using sterile forceps to remove the tip, excluding the cell culture area, and continue to depress the plunger.

iv. Immediately aspirate the discharge fluid from the chip surface using the seeded tip (avoid contact with the port).

v. Use a pipette to immediately remove any spillage from the chip surface using a seeded tip.

Both the inlet and outlet are flush with the chip surface to remove effluent to prevent hydrostatic flow.

K. Cover the dish and transfer it to the microscope to check the density.

L. After seeding, place the chip in the incubator until the cells attach.

i. A small reservoir (15 ml or 50 ml conical tube cap) is placed inside the dish with PBS to provide humidity to the cells.

ii. Attachment time ranges from 1 to 3 hours depending on the cell type.

After the M. cells have attached, gravity wash the chip with warm medium by gently washing the medium through the channels.

N. Place the chip back in the incubator until ready to move to the next step.

Example 4

This example provides one embodiment of a protocol for seeding cells inside a chip in the lower channel (which is horizontally oriented unless otherwise indicated). This example assumes aseptic technique and a sterile environment.

It should be noted that although some cells require very specific seeding conditions, generally optimal seeding density is achieved when cells are closely spaced in a planar monolayer. From this separation, most primary cells will attach and spread into the confluent monolayer.

See “Gravity Washing” below. This means a) placing a drop (bolus) of medium (100uL) onto the port on one side of the channel, making sure no air bubbles are introduced within the port itself, and b) flowing it through the chip, always leaving the outlet port. It involves aspirating excess medium from.

A. Place the dish containing the chips in the hood and aspirate excess medium on the chip surface (handle the chip carriers only by their wings, keep the chip carriers flat and do not pick them up! This will break the chip culture membrane). This will ensure uniform distribution of cells).

B. Gently aspirate the cell suspension before seeding each chip.

C. Pipette 20 μl of cell suspension and seed into the lower channel (when the chip is in a horizontal position, the lower channel is the upper right port) (use 1 chip first).

i. Inject 20 ul of cell suspension and continue to depress the pipette plunger (if you see fluid coming out, the injection was successful; if you see fluid coming out of the same injection port, the tip is not injected properly and see step ii (repeat).

ii. To remove the pipette tip, gently press the chip body using sterile forceps to remove the tip, excluding the cell culture area, and continue to press the plunger.

iii. Immediately aspirate the discharge fluid from the chip surface using the seeded tip (avoid contact with the port).

iv. Both the inlet and outlet are flush with the chip surface to remove effluent to prevent hydrostatic flow.

D. Cover the dish and transfer it to the microscope to check the density.

E. After seeding, invert the chip inside the dish and place the chip in the incubator until the cells adhere underneath the membrane.

i. Attachment time ranges from 1 to 3 hours depending on the cell type.

ii. A small reservoir (15 ml or 50 ml conical tube cap) is placed inside the dish with PBS to provide humidity to the cells.

F. After the cells have attached, invert the chip again and gravity wash the chip with warm medium by gently injecting medium through the channel.

G. Place the chip back in the incubator until ready to move to the next step (cells can be cultured on the chip under static conditions until ready to be connected to a perfusion manifold for flow conditions).

i. Aspirate old media from the chip surface.

ii. Every day, gravity wash the chip with warm medium by gently injecting medium through the channel, 200 ul each for the upper and lower channels, and drop medium from the injection port.

iii. A small reservoir (15 ml or 50 ml conical tube cap) is placed inside the dish with PBS to provide humidity to the cells.

Example 5

In this example, one embodiment of a protocol for making perfusion disposables or “pods” is provided. This assumes aseptic technique and a sterile environment.

A. Pre-warm the medium to 37°C.

B. Transfer the warm medium to the biohood.

C. Aliquot the required amount +5% into a 50 mL conical tube.

D. Sterilize one SteriFlip vacuum filter for each tube of medium and transfer to the hood.

i. Remove the SteriFlip from its packaging and connect it to a 50 mL medium tube.

ii. Inside the hood, connect to the vacuum and invert.

iii. Vacuum degas for at least 15 minutes using a timer.

E. Prepare the correct number of PODs (based on the number of executable chips).

F. Sterilize emulate nests and trays with ethanol and transfer them to the hood.

G. Sterilize one packaged Pod for each viable chip with ethanol and transfer to the hood (always hold only the edges of the POD with your thumb and middle finger, keeping the lid of the POD on top, and holding the POD simultaneously with your index finger). (flatten using .

H. Remove reservoir cover and add medium. This should produce a drop suitable for drop-to-drop engagement of the POD and chip.

i. Inlet reservoir: Fill 1-3 ml (minimum 1 ml).

ii. Outlet reservoir: 300 ul

I. Move the seeded chip from the incubator and bring it into the hood.

i. Remove the pipette tips using a light twisting motion and dispose of them.

ii. Add 10-50 μl of medium onto each port using a 200 μl pipette (avoid creating bubbles inside the port). This should produce a drop suitable for drop-to-drop engagement of the POD and chip.

J. Connect the chip + carrier to the POD. This joining process must utilize the drops formed in steps H and I to create drop-to-drop engagement of the POD and chip.

i. With one hand, place your thumb on the locking mechanism and use your index finger and thumb to grasp the chip carrier and firmly pinch the carrier.

ii. With your other hand, hold the Pod around the reservoir with your thumb and middle finger, and place your index finger on the top of the cover to secure it.

iii. Orient the Pod along its internal tracks so you can see “inside.”

iv. While still holding the carrier, align the carrier's feet along the track inside the Pod.

v. Slide the chip carrier into the pod.

vi. Using your thumb against the chip carrier, lightly press the locking mechanism until it slides into place, capturing the chip inside the Pod.

vii. Ensure that each storage cover is positioned correctly on each Pod.

Claims (14)

a) a culture module comprising an actuation assembly configured to move a pressure manifold in relation to a plurality of microfluidic devices, wherein the pressure manifold includes an integrated valve;
b) wherein the plurality of microfluidic devices are detachably connected to and in contact with the pressure manifold.
system. The system of claim 1, wherein the plurality of microfluidic devices is a plurality of perfusion disposables. 3. The system of claim 1 or 2, wherein the integrated valve comprises a Schrader valve. 3. The device of claim 1 or 2, wherein each of the microfluidic devices further comprises a cover assembly comprising a cover having a plurality of ports, the pressure manifold having pressure points corresponding to ports on the cover. Includes a surface,
A system wherein said pressure points on a mating surface of a pressure manifold contact said plurality of ports of a cover assembly. 5. The system of claim 4, wherein the plurality of ports comprises a plurality of through-hole ports. 5. The system of claim 4, wherein the plurality of ports associate with corresponding holes in the strainer and gasket. 5. The system of claim 4, wherein the culture module further comprises a pressure regulator. 8. The system of claim 7, wherein the pressure regulator is configured to apply pressure through the pressure point. 3. The system of claim 1 or 2, wherein the actuating assembly comprises a pneumatic cylinder operably connected to the pressure manifold. 5. The system of claim 4, wherein the bonding surface further comprises an alignment feature configured to align with each of the microfluidic devices. 5. The system of claim 4, wherein the culture module further comprises an elastomeric membrane, and the elastomeric membrane is in contact with the microfluidic device. 3. The system of claim 1 or 2, wherein the culture module is configured to receive one or more trays, each tray comprising a plurality of microfluidic devices. The system according to claim 1 or 2, wherein the culture module further includes a user interface for controlling the culture module. 3. The system of claim 1 or 2, wherein the plurality of microfluidic devices include ports configured to align with the integrated valve.

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